research paper and reflection paper difference

Organizing Your Social Sciences Research Assignments

• Annotated Bibliography
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• Using Visual Aids
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• Types of Structured Group Activities
• Group Project Survival Skills
• Leading a Class Discussion
• Multiple Book Review Essay
• Reviewing Collected Works
• Writing a Case Analysis Paper
• Writing a Case Study
• About Informed Consent
• Writing Field Notes
• Writing a Policy Memo
• Writing a Reflective Paper
• Writing a Research Proposal
• Generative AI and Writing
• Acknowledgments

Reflective writing is a process of identifying, questioning, and critically evaluating course-based learning opportunities, integrated with your own observations, experiences, impressions, beliefs, assumptions, or biases, and which describes how this process stimulated new or creative understanding about the content of the course.

A reflective paper describes and explains in an introspective, first person narrative, your reactions and feelings about either a specific element of the class [e.g., a required reading; a film shown in class] or more generally how you experienced learning throughout the course. Reflective writing assignments can be in the form of a single paper, essays, portfolios, journals, diaries, or blogs. In some cases, your professor may include a reflective writing assignment as a way to obtain student feedback that helps improve the course, either in the moment or for when the class is taught again.

How to Write a Reflection Paper . Academic Skills, Trent University; Writing a Reflection Paper . Writing Center, Lewis University; Critical Reflection . Writing and Communication Centre, University of Waterloo; Tsingos-Lucas et al. "Using Reflective Writing as a Predictor of Academic Success in Different Assessment Formats." American Journal of Pharmaceutical Education 81 (2017): Article 8.

Benefits of Reflective Writing Assignments

As the term implies, a reflective paper involves looking inward at oneself in contemplating and bringing meaning to the relationship between course content and the acquisition of new knowledge . Educational research [Bolton, 2010; Ryan, 2011; Tsingos-Lucas et al., 2017] demonstrates that assigning reflective writing tasks enhances learning because it challenges students to confront their own assumptions, biases, and belief systems around what is being taught in class and, in so doing, stimulate student’s decisions, actions, attitudes, and understanding about themselves as learners and in relation to having mastery over their learning. Reflection assignments are also an opportunity to write in a first person narrative about elements of the course, such as the required readings, separate from the exegetic and analytical prose of academic research papers.

Reflection writing often serves multiple purposes simultaneously. In no particular order, here are some of reasons why professors assign reflection papers:

• Enhances learning from previous knowledge and experience in order to improve future decision-making and reasoning in practice . Reflective writing in the applied social sciences enhances decision-making skills and academic performance in ways that can inform professional practice. The act of reflective writing creates self-awareness and understanding of others. This is particularly important in clinical and service-oriented professional settings.
• Allows students to make sense of classroom content and overall learning experiences in relation to oneself, others, and the conditions that shaped the content and classroom experiences . Reflective writing places you within the course content in ways that can deepen your understanding of the material. Because reflective thinking can help reveal hidden biases, it can help you critically interrogate moments when you do not like or agree with discussions, readings, or other aspects of the course.
• Increases awareness of one’s cognitive abilities and the evidence for these attributes . Reflective writing can break down personal doubts about yourself as a learner and highlight specific abilities that may have been hidden or suppressed due to prior assumptions about the strength of your academic abilities [e.g., reading comprehension; problem-solving skills]. Reflective writing, therefore, can have a positive affective [i.e., emotional] impact on your sense of self-worth.
• Applying theoretical knowledge and frameworks to real experiences . Reflective writing can help build a bridge of relevancy between theoretical knowledge and the real world. In so doing, this form of writing can lead to a better understanding of underlying theories and their analytical properties applied to professional practice.
• Reveals shortcomings that the reader will identify . Evidence suggests that reflective writing can uncover your own shortcomings as a learner, thereby, creating opportunities to anticipate the responses of your professor may have about the quality of your coursework. This can be particularly productive if the reflective paper is written before final submission of an assignment.
• Helps students identify their tacit [a.k.a., implicit] knowledge and possible gaps in that knowledge . Tacit knowledge refers to ways of knowing rooted in lived experience, insight, and intuition rather than formal, codified, categorical, or explicit knowledge. In so doing, reflective writing can stimulate students to question their beliefs about a research problem or an element of the course content beyond positivist modes of understanding and representation.
• Encourages students to actively monitor their learning processes over a period of time . On-going reflective writing in journals or blogs, for example, can help you maintain or adapt learning strategies in other contexts. The regular, purposeful act of reflection can facilitate continuous deep thinking about the course content as it evolves and changes throughout the term. This, in turn, can increase your overall confidence as a learner.
• Relates a student’s personal experience to a wider perspective . Reflection papers can help you see the big picture associated with the content of a course by forcing you to think about the connections between scholarly content and your lived experiences outside of school. It can provide a macro-level understanding of one’s own experiences in relation to the specifics of what is being taught.
• If reflective writing is shared, students can exchange stories about their learning experiences, thereby, creating an opportunity to reevaluate their original assumptions or perspectives . In most cases, reflective writing is only viewed by your professor in order to ensure candid feedback from students. However, occasionally, reflective writing is shared and openly discussed in class. During these discussions, new or different perspectives and alternative approaches to solving problems can be generated that would otherwise be hidden. Sharing student's reflections can also reveal collective patterns of thought and emotions about a particular element of the course.

Bolton, Gillie. Reflective Practice: Writing and Professional Development . London: Sage, 2010; Chang, Bo. "Reflection in Learning." Online Learning 23 (2019), 95-110; Cavilla, Derek. "The Effects of Student Reflection on Academic Performance and Motivation." Sage Open 7 (July-September 2017): 1–13; Culbert, Patrick. “Better Teaching? You Can Write On It “ Liberal Education (February 2022); McCabe, Gavin and Tobias Thejll-Madsen. The Reflection Toolkit . University of Edinburgh; The Purpose of Reflection . Introductory Composition at Purdue University; Practice-based and Reflective Learning . Study Advice Study Guides, University of Reading; Ryan, Mary. "Improving Reflective Writing in Higher Education: A Social Semiotic Perspective." Teaching in Higher Education 16 (2011): 99-111; Tsingos-Lucas et al. "Using Reflective Writing as a Predictor of Academic Success in Different Assessment Formats." American Journal of Pharmaceutical Education 81 (2017): Article 8; What Benefits Might Reflective Writing Have for My Students? Writing Across the Curriculum Clearinghouse; Rykkje, Linda. "The Tacit Care Knowledge in Reflective Writing: A Practical Wisdom." International Practice Development Journal 7 (September 2017): Article 5; Using Reflective Writing to Deepen Student Learning . Center for Writing, University of Minnesota.

How to Approach Writing a Reflection Paper

Thinking About Reflective Thinking

Educational theorists have developed numerous models of reflective thinking that your professor may use to frame a reflective writing assignment. These models can help you systematically interpret your learning experiences, thereby ensuring that you ask the right questions and have a clear understanding of what should be covered. A model can also represent the overall structure of a reflective paper. Each model establishes a different approach to reflection and will require you to think about your writing differently. If you are unclear how to fit your writing within a particular reflective model, seek clarification from your professor. There are generally two types of reflective writing assignments, each approached in slightly different ways.

1.  Reflective Thinking about Course Readings

This type of reflective writing focuses on thoughtfully thinking about the course readings that underpin how most students acquire new knowledge and understanding about the subject of a course. Reflecting on course readings is often assigned in freshmen-level, interdisciplinary courses where the required readings examine topics viewed from multiple perspectives and, as such, provide different ways of analyzing a topic, issue, event, or phenomenon. The purpose of reflective thinking about course readings in the social and behavioral sciences is to elicit your opinions, beliefs, and feelings about the research and its significance. This type of writing can provide an opportunity to break down key assumptions you may have and, in so doing, reveal potential biases in how you interpret the scholarship.

If you are assigned to reflect on course readings, consider the following methods of analysis as prompts that can help you get started :

• Examine carefully the main introductory elements of the reading, including the purpose of the study, the theoretical framework being used to test assumptions, and the research questions being addressed. Think about what ideas stood out to you. Why did they? Were these ideas new to you or familiar in some way based on your own lived experiences or prior knowledge?
• Develop your ideas around the readings by asking yourself, what do I know about this topic? Where does my existing knowledge about this topic come from? What are the observations or experiences in my life that influence my understanding of the topic? Do I agree or disagree with the main arguments, recommended course of actions, or conclusions made by the author(s)? Why do I feel this way and what is the basis of these feelings?
• Make connections between the text and your own beliefs, opinions, or feelings by considering questions like, how do the readings reinforce my existing ideas or assumptions? How the readings challenge these ideas or assumptions? How does this text help me to better understand this topic or research in ways that motivate me to learn more about this area of study?

2.  Reflective Thinking about Course Experiences

This type of reflective writing asks you to critically reflect on locating yourself at the conceptual intersection of theory and practice. The purpose of experiential reflection is to evaluate theories or disciplinary-based analytical models based on your introspective assessment of the relationship between hypothetical thinking and practical reality; it offers a way to consider how your own knowledge and skills fit within professional practice. This type of writing also provides an opportunity to evaluate your decisions and actions, as well as how you managed your subsequent successes and failures, within a specific theoretical framework. As a result, abstract concepts can crystallize and become more relevant to you when considered within your own experiences. This can help you formulate plans for self-improvement as you learn.

If you are assigned to reflect on your experiences, consider the following questions as prompts to help you get started :

• Contextualize your reflection in relation to the overarching purpose of the course by asking yourself, what did you hope to learn from this course? What were the learning objectives for the course and how did I fit within each of them? How did these goals relate to the main themes or concepts of the course?
• Analyze how you experienced the course by asking yourself, what did I learn from this experience? What did I learn about myself? About working in this area of research and study? About how the course relates to my place in society? What assumptions about the course were supported or refuted?
• Think introspectively about the ways you experienced learning during the course by asking yourself, did your learning experiences align with the goals or concepts of the course? Why or why do you not feel this way? What was successful and why do you believe this? What would you do differently and why is this important? How will you prepare for a future experience in this area of study?

NOTE: If you are assigned to write a journal or other type of on-going reflection exercise, a helpful approach is to reflect on your reflections by re-reading what you have already written. In other words, review your previous entries as a way to contextualize your feelings, opinions, or beliefs regarding your overall learning experiences. Over time, this can also help reveal hidden patterns or themes related to how you processed your learning experiences. Consider concluding your reflective journal with a summary of how you felt about your learning experiences at critical junctures throughout the course, then use these to write about how you grew as a student learner and how the act of reflecting helped you gain new understanding about the subject of the course and its content.

ANOTHER NOTE: Regardless of whether you write a reflection paper or a journal, do not focus your writing on the past. The act of reflection is intended to think introspectively about previous learning experiences. However, reflective thinking should document the ways in which you progressed in obtaining new insights and understandings about your growth as a learner that can be carried forward in subsequent coursework or in future professional practice. Your writing should reflect a furtherance of increasing personal autonomy and confidence gained from understanding more about yourself as a learner.

Structure and Writing Style

There are no strict academic rules for writing a reflective paper. Reflective writing may be assigned in any class taught in the social and behavioral sciences and, therefore, requirements for the assignment can vary depending on disciplinary-based models of inquiry and learning. The organization of content can also depend on what your professor wants you to write about or based on the type of reflective model used to frame the writing assignment. Despite these possible variations, below is a basic approach to organizing and writing a good reflective paper, followed by a list of problems to avoid.

Pre-flection

In most cases, it's helpful to begin by thinking about your learning experiences and outline what you want to focus on before you begin to write the paper. This can help you organize your thoughts around what was most important to you and what experiences [good or bad] had the most impact on your learning. As described by the University of Waterloo Writing and Communication Centre, preparing to write a reflective paper involves a process of self-analysis that can help organize your thoughts around significant moments of in-class knowledge discovery.

• Using a thesis statement as a guide, note what experiences or course content stood out to you , then place these within the context of your observations, reactions, feelings, and opinions. This will help you develop a rough outline of key moments during the course that reflect your growth as a learner. To identify these moments, pose these questions to yourself: What happened? What was my reaction? What were my expectations and how were they different from what transpired? What did I learn?
• Critically think about your learning experiences and the course content . This will help you develop a deeper, more nuanced understanding about why these moments were significant or relevant to you. Use the ideas you formulated during the first stage of reflecting to help you think through these moments from both an academic and personal perspective. From an academic perspective, contemplate how the experience enhanced your understanding of a concept, theory, or skill. Ask yourself, did the experience confirm my previous understanding or challenge it in some way. As a result, did this highlight strengths or gaps in your current knowledge? From a personal perspective, think introspectively about why these experiences mattered, if previous expectations or assumptions were confirmed or refuted, and if this surprised, confused, or unnerved you in some way.
• Analyze how these experiences and your reactions to them will shape your future thinking and behavior . Reflection implies looking back, but the most important act of reflective writing is considering how beliefs, assumptions, opinions, and feelings were transformed in ways that better prepare you as a learner in the future. Note how this reflective analysis can lead to actions you will take as a result of your experiences, what you will do differently, and how you will apply what you learned in other courses or in professional practice.

Basic Structure and Writing Style

Reflective Background and Context

The first part of your reflection paper should briefly provide background and context in relation to the content or experiences that stood out to you. Highlight the settings, summarize the key readings, or narrate the experiences in relation to the course objectives. Provide background that sets the stage for your reflection. You do not need to go into great detail, but you should provide enough information for the reader to understand what sources of learning you are writing about [e.g., course readings, field experience, guest lecture, class discussions] and why they were important. This section should end with an explanatory thesis statement that expresses the central ideas of your paper and what you want the readers to know, believe, or understand after they finish reading your paper.

Reflective Interpretation

Drawing from your reflective analysis, this is where you can be personal, critical, and creative in expressing how you felt about the course content and learning experiences and how they influenced or altered your feelings, beliefs, assumptions, or biases about the subject of the course. This section is also where you explore the meaning of these experiences in the context of the course and how you gained an awareness of the connections between these moments and your own prior knowledge.

Guided by your thesis statement, a helpful approach is to interpret your learning throughout the course with a series of specific examples drawn from the course content and your learning experiences. These examples should be arranged in sequential order that illustrate your growth as a learner. Reflecting on each example can be done by: 1)  introducing a theme or moment that was meaningful to you, 2) describing your previous position about the learning moment and what you thought about it, 3) explaining how your perspective was challenged and/or changed and why, and 4) introspectively stating your current or new feelings, opinions, or beliefs about that experience in class.

It is important to include specific examples drawn from the course and placed within the context of your assumptions, thoughts, opinions, and feelings. A reflective narrative without specific examples does not provide an effective way for the reader to understand the relationship between the course content and how you grew as a learner.

Reflective Conclusions

The conclusion of your reflective paper should provide a summary of your thoughts, feelings, or opinions regarding what you learned about yourself as a result of taking the course. Here are several ways you can frame your conclusions based on the examples you interpreted and reflected on what they meant to you. Each example would need to be tied to the basic theme [thesis statement] of your reflective background section.

• Your reflective conclusions can be described in relation to any expectations you had before taking the class [e.g., “I expected the readings to not be relevant to my own experiences growing up in a rural community, but the research actually helped me see that the challenges of developing my identity as a child of immigrants was not that unusual...”].
• Your reflective conclusions can explain how what you learned about yourself will change your actions in the future [e.g., “During a discussion in class about the challenges of helping homeless people, I realized that many of these people hate living on the street but lack the ability to see a way out. This made me realize that I wanted to take more classes in psychology...”].
• Your reflective conclusions can describe major insights you experienced a critical junctures during the course and how these moments enhanced how you see yourself as a student learner [e.g., "The guest speaker from the Head Start program made me realize why I wanted to pursue a career in elementary education..."].
• Your reflective conclusions can reconfigure or reframe how you will approach professional practice and your understanding of your future career aspirations [e.g.,, "The course changed my perceptions about seeking a career in business finance because it made me realize I want to be more engaged in customer service..."]
• Your reflective conclusions can explore any learning you derived from the act of reflecting itself [e.g., “Reflecting on the course readings that described how minority students perceive campus activities helped me identify my own biases about the benefits of those activities in acclimating to campus life...”].

NOTE: The length of a reflective paper in the social sciences is usually less than a traditional research paper. However, don’t assume that writing a reflective paper is easier than writing a research paper. A well-conceived critical reflection paper often requires as much time and effort as a research paper because you must purposeful engage in thinking about your learning in ways that you may not be comfortable with or used to. This is particular true while preparing to write because reflective papers are not as structured as a traditional research paper and, therefore, you have to think deliberately about how you want to organize the paper and what elements of the course you want to reflect upon.

ANOTHER NOTE: Do not limit yourself to using only text in reflecting on your learning. If you believe it would be helpful, consider using creative modes of thought or expression such as, illustrations, photographs, or material objects that reflects an experience related to the subject of the course that was important to you [e.g., like a ticket stub to a renowned speaker on campus]. Whatever non-textual element you include, be sure to describe the object's relevance to your personal relationship to the course content.

Problems to Avoid

A reflective paper is not a “mind dump” . Reflective papers document your personal and emotional experiences and, therefore, they do not conform to rigid structures, or schema, to organize information. However, the paper should not be a disjointed, stream-of-consciousness narrative. Reflective papers are still academic pieces of writing that require organized thought, that use academic language and tone , and that apply intellectually-driven critical thinking to the course content and your learning experiences and their significance.

A reflective paper is not a research paper . If you are asked to reflect on a course reading, the reflection will obviously include some description of the research. However, the goal of reflective writing is not to present extraneous ideas to the reader or to "educate" them about the course. The goal is to share a story about your relationship with the learning objectives of the course. Therefore, unlike research papers, you are expected to write from a first person point of view which includes an introspective examination of your own opinions, feelings, and personal assumptions.

A reflection paper is not a book review . Descriptions of the course readings using your own words is not a reflective paper. Reflective writing should focus on how you understood the implications of and were challenged by the course in relation to your own lived experiences or personal assumptions, combined with explanations of how you grew as a student learner based on this internal dialogue. Remember that you are the central object of the paper, not the research materials.

A reflective paper is not an all-inclusive meditation. Do not try to cover everything. The scope of your paper should be well-defined and limited to your specific opinions, feelings, and beliefs about what you determine to be the most significant content of the course and in relation to the learning that took place. Reflections should be detailed enough to covey what you think is important, but your thoughts should be expressed concisely and coherently [as is true for any academic writing assignment].

Critical Reflection . Writing and Communication Centre, University of Waterloo; Critical Reflection: Journals, Opinions, & Reactions . University Writing Center, Texas A&M University; Connor-Greene, Patricia A. “Making Connections: Evaluating the Effectiveness of Journal Writing in Enhancing Student Learning.” Teaching of Psychology 27 (2000): 44-46; Good vs. Bad Reflection Papers , Franklin University; Dyment, Janet E. and Timothy S. O’Connell. "The Quality of Reflection in Student Journals: A Review of Limiting and Enabling Factors." Innovative Higher Education 35 (2010): 233-244: How to Write a Reflection Paper . Academic Skills, Trent University; Amelia TaraJane House. Reflection Paper . Cordia Harrington Center for Excellence, University of Arkansas; Ramlal, Alana, and Désirée S. Augustin. “Engaging Students in Reflective Writing: An Action Research Project.” Educational Action Research 28 (2020): 518-533; Writing a Reflection Paper . Writing Center, Lewis University; McGuire, Lisa, Kathy Lay, and Jon Peters. “Pedagogy of Reflective Writing in Professional Education.” Journal of the Scholarship of Teaching and Learning (2009): 93-107; Critical Reflection . Writing and Communication Centre, University of Waterloo; How Do I Write Reflectively? Academic Skills Toolkit, University of New South Wales Sydney; Reflective Writing . Skills@Library. University of Leeds; Walling, Anne, Johanna Shapiro, and Terry Ast. “What Makes a Good Reflective Paper?” Family Medicine 45 (2013): 7-12; Williams, Kate, Mary Woolliams, and Jane Spiro. Reflective Writing . 2nd edition. London: Red Globe Press, 2020; Yeh, Hui-Chin, Shih-hsien Yang, Jo Shan Fu, and Yen-Chen Shih. “Developing College Students’ Critical Thinking through Reflective Writing.” Higher Education Research and Development (2022): 1-16.

Writing Tip

Focus on Reflecting, Not on Describing

Minimal time and effort should be spent describing the course content you are asked to reflect upon. The purpose of a reflection assignment is to introspectively contemplate your reactions to and feeling about an element of the course. D eflecting the focus away from your own feelings by concentrating on describing the course content can happen particularly if "talking about yourself" [i.e., reflecting] makes you uncomfortable or it is intimidating. However, the intent of reflective writing is to overcome these inhibitions so as to maximize the benefits of introspectively assessing your learning experiences. Keep in mind that, if it is relevant, your feelings of discomfort could be a part of how you critically reflect on any challenges you had during the course [e.g., you realize this discomfort inhibited your willingness to ask questions during class, it fed into your propensity to procrastinate, or it made it difficult participating in groups].

Writing a Reflection Paper . Writing Center, Lewis University; Reflection Paper . Cordia Harrington Center for Excellence, University of Arkansas.

Another Writing Tip

Helpful Videos about Reflective Writing

These two short videos succinctly describe how to approach a reflective writing assignment. They are produced by the Academic Skills department at the University of Melbourne and the Skills Team of the University of Hull, respectively.

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Structure of academic reflections

Guidance on the structure of academic reflections.

Academic reflections or reflective writing completed for assessment often require a clear structure. Contrary to some people’s belief, reflection is not just a personal diary talking about your day and your feelings.

Both the language and the structure are important for academic reflective writing. For the structure you want to mirror an academic essay closely. You want an introduction, a main body, and a conclusion.

Academic reflection will require you to both describe the context, analyse it, and make conclusions. However, there is not one set of rules for the proportion of your reflection that should be spent describing the context, and what proportion should be spent on analysing and concluding. That being said, as learning tends to happen when analysing and synthesising rather than describing, a good rule of thumb is to describe just enough such that the reader understands your context.

Example structure for academic reflections

Below is an example of how you might structure an academic reflection if you were given no other guidance and what each section might contain.  Remember this is only a suggestion and you must consider what is appropriate for the task at hand and for you yourself.

Introduction

Identifies and introduces your experience or learning

• This can be a critical incident
• This can be the reflective prompt you were given
• A particular learning you have gained

When structuring your academic reflections it might make sense to start with what you have learned and then use the main body to evidence that learning, using specific experiences and events. Alternatively, start with the event and build up your argument. This is a question of personal preference – if you aren’t given explicit guidance you can ask the assessor if they have a preference, however both can work.

Highlights why it was important

• This can be suggesting why this event was important for the learning you gained
• This can be why the learning you gained will benefit you or why you appreciate it in your context

You might find that it is not natural to highlight the importance of an event before you have developed your argument for what you gained from it. It can be okay not to explicitly state the importance in the introduction, but leave it to develop throughout your reflection.

Outline key themes that will appear in the reflection (optional – but particularly relevant when answering a reflective prompt or essay)

• This can be an introduction to your argument, introducing the elements that you will explore, or that builds to the learning you have already gained.

This might not make sense if you are reflecting on a particular experience, but is extremely valuable if you are answering a reflective prompt or writing an essay that includes multiple learning points. A type of prompt or question that could particularly benefit from this would be ‘Reflect on how the skills and theory within this course have helped you meet the benchmark statements of your degree’

It can be helpful to explore one theme/learning per paragraph.

Explore experiences

• You should highlight and explore the experience you introduced in the introduction
• If you are building toward answering a reflective prompt, explore each relevant experience.

As reflection is centred around an individual’s personal experience, it is very important to make experiences a main component of reflection. This does not mean that the majority of the reflective piece should be on describing an event – in fact you should only describe enough such that the reader can follow your analysis.

Analyse and synthesise

• You should analyse each of your experiences and from them synthesise new learning

Depending on the requirements of the assessment, you may need to use theoretical literature in your analysis. Theoretical literature is a part of perspective taking which is relevant for reflection, and will happen as a part of your analysis.  

Restate or state your learning

• Make a conclusion based on your analysis and synthesis.
• If you have many themes in your reflection, it can be helpful to restate them here.

Plan for the future

• Highlight and discuss how your new-found learnings will influence your future practice

Answer the question or prompt (if applicable)

• If you are answering an essay question or reflective prompt, make sure that your conclusion provides a succinct response using your main body as evidence.  

Using a reflective model to structure academic reflections

You might recognise that most reflective models mirror this structure; that is why a lot of the reflective models can be really useful to structure reflective assignments. Models are naturally structured to focus on a single experience – if the assignment requires you to focus on multiple experiences, it can be helpful to simply repeat each step of a model for each experience.

One difference between the structure of reflective writing and the structure of models is that sometimes you may choose to present your learning in the introduction of a piece of writing, whereas models (given that they support working through the reflective process) will have learning appearing at later stages.

However, generally structuring a piece of academic writing around a reflective model will ensure that it involves the correct components, reads coherently and logically, as well as having an appropriate structure.

Reflective journals/diaries/blogs and other pieces of assessed reflection

The example structure above works particularly well for formal assignments such as reflective essays and reports.  Reflective journal/blogs and other pieces of assessed reflections tend to be less formal both in language and structure, however you can easily adapt the structure for journals and other reflective assignments if you find that helpful.

That is, if you are asked to produce a reflective journal with multiple entries it will most often (always check with the person who issued the assignment) be a successful journal if each entry mirrors the structure above and the language highlighted in the section on academic language. However, often you can be less concerned with form when producing reflective journals/diaries.

When producing reflective journals, it is often okay to include your original reflection as long as you are comfortable with sharing the content with others, and that the information included is not too personal for an assessor to read.

Developed from:

Ryan, M., 2011. Improving reflective writing in higher education: a social semiotic perspective. Teaching in Higher Education, 16(1), 99-111.

University of Portsmouth, Department for Curriculum and Quality Enhancement (date unavailable). Reflective Writing: a basic introduction [online].  Portsmouth: University of Portsmouth.

Queen Margaret University, Effective Learning Service (date unavailable).  Reflection. [online].  Edinburgh: Queen Margaret University.

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How to Write a Reflection Paper: Definition, Outline, Steps & Examples

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Reflection paper is an opportunity to look at a topic, concept or event and analyze it. It can involve personal introspection, observations of a particular situation or event, and even critical analysis of other works. Students should share their emotions, opinions, and reflections, exploring how the subject matter has impacted their thinking and personal growth. Unlike other types of essays, a reflection paper is usually written in the first person. 

Whether your teacher assigns an  internship reflection paper or any other type of a reflection paper, don't write about the image in the mirror. On the contrary,  study your thoughts on a given topic. Most students first encounter this type of writing when describing how they spent summer.  However, this type of academic writing can cover much more. In this article, you will find everything you need to know about this type of academic piece!

What Is a Reflection Paper: A Detailed Definition

Reflection paper refers to a type of academic writing where you should analyze your personal life, and explore specific ideas of how your changes, development, or growth turned out.  Consider this piece like diary entries. Except that others will be reading them. So it should have consistency, reasonable structure, and be easy to understand. In this respect, this work is very similar to any other academic assignment. Simply put, a reflective paper is a critique of life experiences. And with proper guidance, it is not very difficult to compose. Moreover, there are different types of reflection papers . After all, you can reflect on different things, not only your own experience. These types are:

• Educational reflection paper In this type of work you must write feedback about a book, movie, or seminar you attended.
• Professional paper It is usually written by people who study or work in education or psychology.
• Personal paper It goes without saying that this type is all about your own feelings and thoughts on a particular topic.

Reflection Paper Format: Which One to Choose

Reflection paper format can vary slightly depending on who your audience is. It is not uncommon that your paper format will be assigned specifically by your professor. However, some essential structural elements are typical for MLA, APA, or Chicago style formatting. These include introduction, body, and conclusion. You can find more information on paper formats in our blog. As always, paper writers for hire at StudyCrumb are at your hand 24/7.

How to Start a Reflection Paper: Guidelines

Here, we will explain how to begin a reflection paper. Working on how to start a body paragraph , review criteria for evaluation. This first step will help you concentrate on what is required. In the beginning, summarize brief information with no spoilers. Then professionally explain what thoughts you (if it is a personal paper) or a writer (if educational or professional paper) touch upon. But still, remember that essays should be written in first person and focus on "you."

Reflection Paper Outline

The best reflection paper outline consists of an introduction that attracts attention. After introduction, the plan includes the main body and, finally, conclusions. Adherence to this structure will allow you to clearly express your thinking. The detailed description of each part is right below.

Reflection Paper Introduction: Start With Hook

Reflection paper introduction starts with a hook. Find a way to intrigue your reader and make them interested in your assignment before they even read it. Also, you should briefly and informatively describe the background and thesis statement. Make it clear and concise, so neither you nor your reader would get confused later. Don't forget to state what it is you're writing about: an article, a personal experience, a book, or something else.

Number of Body Paragraphs in a Reflective Paper

Reflective paper body paragraphs explain how your thinking has changed according to something. Don't only share changes but also provide examples as supporting details. For example, if you discuss how to become more optimistic, describe what led to this change. Examples serve as supporting structure of your assignment. They are similar to evidence in, say, an argumentative essay.  Keep in mind that your work doesn't have to be disengaged and aloof. It is your own experience you're sharing, after all.

How to End a Reflection Paper

In the short reflection paper conclusion, you summarize the thesis and personal experience. It's fascinating that in this academic work, you can reflect forward or backward on your experience. In the first case, you share what role the essay plays in your future. In the second case, you focus more on the past. You acknowledge the impact that the essay's story has on your life. Reflect on how you changed bit by bit, or, maybe, grew as a person. Perhaps, you have witnessed something so fascinating it changed your outlook on certain aspects of your life. This is how to write conclusion in research paper in the best way possible.

How to Write Reflection Paper: Full Step-By-Step Guide

Writing reflection paper could be initiated by the teacher at college. Or we can even do it by ourselves to challenge our evaluation skills and see how we have changed. In any case, it's not an issue anymore since we've prepared a super handy guide. Just follow it step by step, and you will be amazed at the result.

Step 1. Answer the Main Questions Before Writing a Reflection Paper

A reflection paper means you should provide your thoughts on the specific topic and cover some responses. So before writing, research the information you want to apply and note every idea. If you're writing an educational or professional paper ask yourself several questions, for example:

• What was my viewpoint before reading this book?
• How do I consider this situation now?
• What does this book teach me?

If your goal is to reflect on personal experience, you can start with asking questions like:

• What was your viewpoint before the experience?
• How did this experience change your viewpoint?

The more details you imagine, the better you can answer these questions. 

Step 2. Identify the Main Theme of Your Reflection Paper

Reflection papers' suggested topics can be varied. Generally, it could be divided into four main categories to discuss:

• Articles or books.
• Social events.
• Persons or famous individuals.
• Personal experiences.

In any case, it's good to show your own attitude to a topic, and that it affects yourself. It is also suited to write about your own negative experiences and mistakes. You need to show how you overcame some obstacle, or maybe you're still dealing with the consequences of your choices. Consider what you learnt through this experience, and how it makes you who you are now.

Step 3. Summarize the Material for Reflection Paper

At this step of reflective paper, you can wait for inspiration and brainstorm. Don't be afraid of a blank sheet. Carefully read the topic suggested for the essay. Think about associations, comparisons, facts that immediately come to mind. If the teacher recommended particular literature, find it. If not, check the previous topic's background. Remember how to quote a quote that you liked, but be sure to indicate its author and source. Think of relevant examples or look for statistics, and analyze them. Just start drafting a summary of everything you know regarding this topic. And keep in mind, that main task is to describe your own thoughts and feelings.

Step 4. Analyze Main Aspects of Reflection Paper

A whole reflection paper's meaning lies in putting theory and your experience together. So fill in different ideas in your piece step by step until you realize there's enough material. If you may find some particular quotes, you should focus on your viewpoint and feelings. Who knows, maybe there is some relatable literature (or video material) that can highlight your idea and make it sound more engaging?

The Best Tips on Writing a Reflection Paper

We prepared tips on writing reflection paper to help you find evidence that your work was excellently done! Some, of course, go without saying. Edit your piece for some time after writing, when you cooled down a bit. Pay attention to whether your readers would be interested in this material. Write about things that not only are interesting for you, but have a sufficient amount of literature to read about. Below you will find more tips on various types of writing!

Tips on Writing a Critical Reflection Paper

Role of a critical reflection paper is to change your opinion about a particular subject, thus changing your behaviour. You may ask yourself how your experience could have been improved and what you have to do in order to achieve that. It could be one of the most challenging tasks if you choose the wrong topic. Usually, such works are written at the subject's culmination. This requires intensive, clear, evaluative, and critical context thinking.

• Describe experience in detail.
• Study topic of work well.
• Provide an in-depth analysis.
• Tell readers how this experience changed you.
• Find out how it will affect your future.

Tips on Writing a Course Reflection Paper

Course reflection paper is basically a personal experience of how a course at your college (or university) has affected you. It requires description and title of course, first of all. 

• Clearly write information you discussed, how class went, and reasons you attended it.
• Identify basic concepts, theories and instructions studied. Then interpret them using real-life examples.
• Evaluate relevance and usefulness of course.

How to Write a Reflection Paper on a Book

A reflection paper on a book introduces relevant author's and piece's information. Focus on main characters. Explain what problems are revealed in work, their consequences, and their effectiveness. Share your experience or an example from your personal life. 

How to Write a Reflection Paper on a Project

Main point of a reflection paper on a project is to share your journey during a process. It has the same structure and approach as previous works. Tell all about the obstacles that you needed to overcome. Explain what it took to overcome them. Share your thoughts! Compare your experience with what could have been if there were another approach. But the main task here is to support the pros or cons of the path you've taken. Suggest changes and recognize complexity or relevance to the real world.

How to Write a Reflection Paper on an Interview

A reflection paper on an interview requires a conclusion already in your introduction.

• Introduce the person.
• Then emphasize known points of view, focusing on arguments.
• Later, express what you like or dislike about this idea.

It is always a good idea to brainstorm and research certain interview questions you're planning on discussing with a person. Create an outline of how you want your interview to go. Also, don't digress from a standard 5-paragraph structure, keep your essay simple. You may need a guide on how to write a response paper as well. There is a blog with detailed steps on our website.

Reflection Paper Example

Before we've explained all fundamental basics to you. Now let's look at a reflection paper example. In this file, you'll find a visual structure model and way of thinking expressed.

Reflection Paper: Main Takeaways

A reflection paper is your flow of thoughts in an organized manner concerning any research paper topics . Format is similar to any other academic work. Start with a strong introduction, develop the main body, and end with conclusions. With the help of our article, you can write this piece only in 4 steps.

Our academic assistants are up for the task! Just pick a twitter to your liking, send them your paper requirements and they'll write your reflection paper for you!

Frequently Asked Questions About Reflection Paper

1. how long should a reflection paper be.

A reflection paper must be between 300 and 750 words. Still, it always depends on your previous research and original task requirements. The main task is to cover all essential questions in the narrative flow. So don't stick directly to the work's volume.

2. Do reflection papers need a cover sheet or title page?

A cover sheet or title page isn't necessary for reflection papers. But your teacher may directly require this page. Then you should include a front-page and format it accordingly.

3. Do I need to use citations and references with a reflection paper?

No, usually, you don't have to cite in your reflection paper. It should be only your personal experience and viewpoint. But in some cases, your teacher may require you to quote a certain number of sources. It's necessary that the previous research was completed, so check it beforehand.

4. What is the difference between a reflection paper and a reaction paper?

The research paper definition differs from reaction paper. Basically, the main point is in-depth of discussion. In the first case, you must fully describe how something affected you. While in the second one, it is just asked to provide a simple observation.

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Practical Reflective Assessments

Consider developing reflective partners inside or outside your department or outside the library

Utilize Daily Reflection Log - What frustrations did you encounter? - What new question has arisen in your research? - What was an important discovery today?

Engage in Post-Research Reflection - What went well with this project? - What could have gone better? - How could you make the research experience better next time?

                                    Donham, J.

“We do not learn from experience...we learn from reflecting on experience.”

- John Dewey

Reflective critique is an essential practice during a research project. It should be woven throughout, involving every step of the process from doing a literature search, to choosing a topic, to formulating your methodology, to engaging in the peer review process, and most importantly, as a final step upon completion of a research project.   This page is designed to assist you in developing your abilities to employ reflective critique, so you will know what mindset and activities can assure you that you are engaging with your project both critically and reflectively.

Brooks, S. V., & Bigelow, S. (n.d.). Learning and teaching in action. Health Information and Libraries Journal , 32 (4), 332–338.  https://doi.org/10.1111/hir.12123

Di Stefano, G., Gino, F., Pisano, G. P., & Staats, B. R. (2014). Learning by thinking: How reflection aids performance. SSRN Electronic Journal .  https://doi.org/10.2139/ssrn.2414478

"We find that individuals who are given time to reflect on a task improve their performance at a greater rate than those who are given the same amount of time to practice with the same task. Our results also show that if individuals themselves are given the choice to either reflect or practice, they prefer to allocate their time to gaining more experience with the task – to the detriment of their learning." [from article abstract]

Donham, J. (February 2010). Creating personal learning through self-assessment. Teacher Librarian 37(3), 14-21. http://ezproxy.fiu.edu/login?url=http://search.ebscohost.com/login.aspx?direct=true&db=edsgea&AN=edsgcl.222556198&site=eds-live

Dymarz, A. & Cameron, A. (2015). Self-assessment in librarianship: An exploratory study of current practices and future possibilities. Partnership: The Canadian Journal of Library and Information Practice and Research, 10 (1) , 1-19.  https://doi.org/10.21083/partnership.v10i1.3332

Forrest, Margaret E. S., (n.d). Learning and teaching in action: On becoming a critically reflective practitioner. Health Information and Libraries Journal , 25, 229-232.  https://doi.org/10.1111/j.1471-1842.2008.00787.x

Graf, A. J., & Harris, B. R. (2016). Reflective assessment: Opportunities and challenges. Reference Services Review, 44 (1), 38-47.  http://dx.doi.org/10.1108/RSR-06-2015-0027

"Adding a reflective layer to assessment can help us more carefully evaluate what it is we profess to value in the first place"

Hampe, N. (2013).  Reflective Practice and Writing: a Guide to Getting Started.  Australian Library and Information Association (ALIA).   http://www.alia.org.au/sites/default/files/documents/Reflective.Practice.Writing.Guide20130409JB.pdf

Hernon, P. & Schwartz, C. (2008). A research study's reflective inquiry, Library & Information Science Research , 30 (3), 164–165.  https://doi.org/10.1016/j.lisr.2008.06.003

"What worked? What didn’t work? Were my/our goals met?"

"Does the scholar:

• critically evaluate their work?
• bring an appropriate breadth of evidence to their critique?
• use evaluation to improve the quality of future work?"

"Reflection helps the individual to learn from experience because of the meaningful nature of the inquiry into that experience."

"One's learning [...] can be facilitated through the advisement of one or more significant individuals with whom to engage in a reflective process about one's own thoughts and behaviors." (p.172)

"The simple idea of stepping back in order to move forward is so often overlooked in cultures of action and progress." (p.169)

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Guide on How to Write a Reflection Paper with Free Tips and Example

A reflection paper is a very common type of paper among college students. Almost any subject you enroll in requires you to express your opinion on certain matters. In this article, we will explain how to write a reflection paper and provide examples and useful tips to make the essay writing process easier.

Reflection papers should have an academic tone yet be personal and subjective. In this paper, you should analyze and reflect upon how an experience, academic task, article, or lecture shaped your perception and thoughts on a subject.

Here is what you need to know about writing an effective critical reflection paper. Stick around until the end of our guide to get some useful writing tips from the writing team at EssayPro — a research paper writing service

What Is a Reflection Paper

A reflection paper is a type of paper that requires you to write your opinion on a topic, supporting it with your observations and personal experiences. As opposed to presenting your reader with the views of other academics and writers, in this essay, you get an opportunity to write your point of view—and the best part is that there is no wrong answer. It is YOUR opinion, and it is your job to express your thoughts in a manner that will be understandable and clear for all readers that will read your paper. The topic range is endless. Here are some examples: whether or not you think aliens exist, your favorite TV show, or your opinion on the outcome of WWII. You can write about pretty much anything.

There are three types of reflection paper; depending on which one you end up with, the tone you write with can be slightly different. The first type is the educational reflective paper. Here your job is to write feedback about a book, movie, or seminar you attended—in a manner that teaches the reader about it. The second is the professional paper. Usually, it is written by people who study or work in education or psychology. For example, it can be a reflection of someone’s behavior. And the last is the personal type, which explores your thoughts and feelings about an individual subject.

However, reflection paper writing will stop eventually with one very important final paper to write - your resume. This is where you will need to reflect on your entire life leading up to that moment. To learn how to list education on resume perfectly, follow the link on our dissertation writing services .

Unlock the potential of your thoughts with EssayPro . Order a reflection paper and explore a range of other academic services tailored to your needs. Dive deep into your experiences, analyze them with expert guidance, and turn your insights into an impactful reflection paper.

Free Reflection Paper Example

Now that we went over all of the essentials about a reflection paper and how to approach it, we would like to show you some examples that will definitely help you with getting started on your paper.

Reflection Paper Format

Reflection papers typically do not follow any specific format. Since it is your opinion, professors usually let you handle them in any comfortable way. It is best to write your thoughts freely, without guideline constraints. If a personal reflection paper was assigned to you, the format of your paper might depend on the criteria set by your professor. College reflection papers (also known as reflection essays) can typically range from about 400-800 words in length.

Here’s how we can suggest you format your reflection paper:

How to Start a Reflection Paper

The first thing to do when beginning to work on a reflection essay is to read your article thoroughly while taking notes. Whether you are reflecting on, for example, an activity, book/newspaper, or academic essay, you want to highlight key ideas and concepts.

You can start writing your reflection paper by summarizing the main concept of your notes to see if your essay includes all the information needed for your readers. It is helpful to add charts, diagrams, and lists to deliver your ideas to the audience in a better fashion.

After you have finished reading your article, it’s time to brainstorm. We’ve got a simple brainstorming technique for writing reflection papers. Just answer some of the basic questions below:

• How did the article affect you?
• How does this article catch the reader’s attention (or does it all)?
• Has the article changed your mind about something? If so, explain how.
• Has the article left you with any questions?
• Were there any unaddressed critical issues that didn’t appear in the article?
• Does the article relate to anything from your past reading experiences?
• Does the article agree with any of your past reading experiences?

Here are some reflection paper topic examples for you to keep in mind before preparing to write your own:

• How my views on rap music have changed over time
• My reflection and interpretation of Moby Dick by Herman Melville
• Why my theory about the size of the universe has changed over time
• How my observations for clinical psychological studies have developed in the last year

The result of your brainstorming should be a written outline of the contents of your future paper. Do not skip this step, as it will ensure that your essay will have a proper flow and appropriate organization.

Another good way to organize your ideas is to write them down in a 3-column chart or table.

Do you want your task look awesome?

If you would like your reflection paper to look professional, feel free to check out one of our articles on how to format MLA, APA or Chicago style

Writing a Reflection Paper Outline

Reflection paper should contain few key elements:

Introduction

Your introduction should specify what you’re reflecting upon. Make sure that your thesis informs your reader about your general position, or opinion, toward your subject.

• State what you are analyzing: a passage, a lecture, an academic article, an experience, etc...)
• Briefly summarize the work.
• Write a thesis statement stating how your subject has affected you.

One way you can start your thesis is to write:

Example: “After reading/experiencing (your chosen topic), I gained the knowledge of…”

Body Paragraphs

The body paragraphs should examine your ideas and experiences in context to your topic. Make sure each new body paragraph starts with a topic sentence.

Your reflection may include quotes and passages if you are writing about a book or an academic paper. They give your reader a point of reference to fully understand your feedback. Feel free to describe what you saw, what you heard, and how you felt.

Example: “I saw many people participating in our weight experiment. The atmosphere felt nervous yet inspiring. I was amazed by the excitement of the event.”

As with any conclusion, you should summarize what you’ve learned from the experience. Next, tell the reader how your newfound knowledge has affected your understanding of the subject in general. Finally, describe the feeling and overall lesson you had from the reading or experience.

There are a few good ways to conclude a reflection paper:

• Tie all the ideas from your body paragraphs together, and generalize the major insights you’ve experienced.
• Restate your thesis and summarize the content of your paper.

We have a separate blog post dedicated to writing a great conclusion. Be sure to check it out for an in-depth look at how to make a good final impression on your reader.

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How to Write a Reflection Paper: Step-by-Step Guide

Step 1: create a main theme.

After you choose your topic, write a short summary about what you have learned about your experience with that topic. Then, let readers know how you feel about your case — and be honest. Chances are that your readers will likely be able to relate to your opinion or at least the way you form your perspective, which will help them better understand your reflection.

For example: After watching a TEDx episode on Wim Hof, I was able to reevaluate my preconceived notions about the negative effects of cold exposure.

Step 2: Brainstorm Ideas and Experiences You’ve Had Related to Your Topic

You can write down specific quotes, predispositions you have, things that influenced you, or anything memorable. Be personal and explain, in simple words, how you felt.

For example: • A lot of people think that even a small amount of carbohydrates will make people gain weight • A specific moment when I struggled with an excess weight where I avoided carbohydrates entirely • The consequences of my actions that gave rise to my research • The evidence and studies of nutritional science that claim carbohydrates alone are to blame for making people obese • My new experience with having a healthy diet with a well-balanced intake of nutrients • The influence of other people’s perceptions on the harm of carbohydrates, and the role their influence has had on me • New ideas I’ve created as a result of my shift in perspective

Step 3: Analyze How and Why These Ideas and Experiences Have Affected Your Interpretation of Your Theme

Pick an idea or experience you had from the last step, and analyze it further. Then, write your reasoning for agreeing or disagreeing with it.

For example, Idea: I was raised to think that carbohydrates make people gain weight.

Analysis: Most people think that if they eat any carbohydrates, such as bread, cereal, and sugar, they will gain weight. I believe in this misconception to such a great extent that I avoided carbohydrates entirely. As a result, my blood glucose levels were very low. I needed to do a lot of research to overcome my beliefs finally. Afterward, I adopted the philosophy of “everything in moderation” as a key to a healthy lifestyle.

For example: Idea: I was brought up to think that carbohydrates make people gain weight. Analysis: Most people think that if they eat any carbohydrates, such as bread, cereal, and sugar, they will gain weight. I believe in this misconception to such a great extent that I avoided carbohydrates entirely. As a result, my blood glucose levels were very low. I needed to do a lot of my own research to finally overcome my beliefs. After, I adopted the philosophy of “everything in moderation” as a key for having a healthy lifestyle.

Step 4: Make Connections Between Your Observations, Experiences, and Opinions

Try to connect your ideas and insights to form a cohesive picture for your theme. You can also try to recognize and break down your assumptions, which you may challenge in the future.

There are some subjects for reflection papers that are most commonly written about. They include:

• Book – Start by writing some information about the author’s biography and summarize the plot—without revealing the ending to keep your readers interested. Make sure to include the names of the characters, the main themes, and any issues mentioned in the book. Finally, express your thoughts and reflect on the book itself.
• Course – Including the course name and description is a good place to start. Then, you can write about the course flow, explain why you took this course, and tell readers what you learned from it. Since it is a reflection paper, express your opinion, supporting it with examples from the course.
• Project – The structure for a reflection paper about a project has identical guidelines to that of a course. One of the things you might want to add would be the pros and cons of the course. Also, mention some changes you might want to see, and evaluate how relevant the skills you acquired are to real life.
• Interview – First, introduce the person and briefly mention the discussion. Touch on the main points, controversies, and your opinion of that person.

Writing Tips

Everyone has their style of writing a reflective essay – and that's the beauty of it; you have plenty of leeway with this type of paper – but there are still a few tips everyone should incorporate.

Before you start your piece, read some examples of other papers; they will likely help you better understand what they are and how to approach yours. When picking your subject, try to write about something unusual and memorable — it is more likely to capture your readers' attention. Never write the whole essay at once. Space out the time slots when you work on your reflection paper to at least a day apart. This will allow your brain to generate new thoughts and reflections.

• Short and Sweet – Most reflection papers are between 250 and 750 words. Don't go off on tangents. Only include relevant information.
• Clear and Concise – Make your paper as clear and concise as possible. Use a strong thesis statement so your essay can follow it with the same strength.
• Maintain the Right Tone – Use a professional and academic tone—even though the writing is personal.
• Cite Your Sources – Try to cite authoritative sources and experts to back up your personal opinions.
• Proofreading – Not only should you proofread for spelling and grammatical errors, but you should proofread to focus on your organization as well. Answer the question presented in the introduction.

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Daniel Parker

is a seasoned educational writer focusing on scholarship guidance, research papers, and various forms of academic essays including reflective and narrative essays. His expertise also extends to detailed case studies. A scholar with a background in English Literature and Education, Daniel’s work on EssayPro blog aims to support students in achieving academic excellence and securing scholarships. His hobbies include reading classic literature and participating in academic forums.

is an expert in nursing and healthcare, with a strong background in history, law, and literature. Holding advanced degrees in nursing and public health, his analytical approach and comprehensive knowledge help students navigate complex topics. On EssayPro blog, Adam provides insightful articles on everything from historical analysis to the intricacies of healthcare policies. In his downtime, he enjoys historical documentaries and volunteering at local clinics.

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• How to Write Discussions and Conclusions

The discussion section contains the results and outcomes of a study. An effective discussion informs readers what can be learned from your experiment and provides context for the results.

What makes an effective discussion?

When you’re ready to write your discussion, you’ve already introduced the purpose of your study and provided an in-depth description of the methodology. The discussion informs readers about the larger implications of your study based on the results. Highlighting these implications while not overstating the findings can be challenging, especially when you’re submitting to a journal that selects articles based on novelty or potential impact. Regardless of what journal you are submitting to, the discussion section always serves the same purpose: concluding what your study results actually mean.

A successful discussion section puts your findings in context. It should include:

• the results of your research,
• a discussion of related research, and
• a comparison between your results and initial hypothesis.

Tip: Not all journals share the same naming conventions.

You can apply the advice in this article to the conclusion, results or discussion sections of your manuscript.

Our Early Career Researcher community tells us that the conclusion is often considered the most difficult aspect of a manuscript to write. To help, this guide provides questions to ask yourself, a basic structure to model your discussion off of and examples from published manuscripts. 

Questions to ask yourself:

• Was my hypothesis correct?
• If my hypothesis is partially correct or entirely different, what can be learned from the results? 
• How do the conclusions reshape or add onto the existing knowledge in the field? What does previous research say about the topic? 
• Why are the results important or relevant to your audience? Do they add further evidence to a scientific consensus or disprove prior studies? 
• How can future research build on these observations? What are the key experiments that must be done? 
• What is the “take-home” message you want your reader to leave with?

How to structure a discussion

Trying to fit a complete discussion into a single paragraph can add unnecessary stress to the writing process. If possible, you’ll want to give yourself two or three paragraphs to give the reader a comprehensive understanding of your study as a whole. Here’s one way to structure an effective discussion:

Writing Tips

While the above sections can help you brainstorm and structure your discussion, there are many common mistakes that writers revert to when having difficulties with their paper. Writing a discussion can be a delicate balance between summarizing your results, providing proper context for your research and avoiding introducing new information. Remember that your paper should be both confident and honest about the results! 

• Read the journal’s guidelines on the discussion and conclusion sections. If possible, learn about the guidelines before writing the discussion to ensure you’re writing to meet their expectations. 
• Begin with a clear statement of the principal findings. This will reinforce the main take-away for the reader and set up the rest of the discussion. 
• Explain why the outcomes of your study are important to the reader. Discuss the implications of your findings realistically based on previous literature, highlighting both the strengths and limitations of the research. 
• State whether the results prove or disprove your hypothesis. If your hypothesis was disproved, what might be the reasons? 
• Introduce new or expanded ways to think about the research question. Indicate what next steps can be taken to further pursue any unresolved questions. 
• If dealing with a contemporary or ongoing problem, such as climate change, discuss possible consequences if the problem is avoided. 
• Be concise. Adding unnecessary detail can distract from the main findings. 

Don’t

• Rewrite your abstract. Statements with “we investigated” or “we studied” generally do not belong in the discussion. 
• Include new arguments or evidence not previously discussed. Necessary information and evidence should be introduced in the main body of the paper. 
• Apologize. Even if your research contains significant limitations, don’t undermine your authority by including statements that doubt your methodology or execution. 
• Shy away from speaking on limitations or negative results. Including limitations and negative results will give readers a complete understanding of the presented research. Potential limitations include sources of potential bias, threats to internal or external validity, barriers to implementing an intervention and other issues inherent to the study design. 
• Overstate the importance of your findings. Making grand statements about how a study will fully resolve large questions can lead readers to doubt the success of the research. 

Snippets of Effective Discussions:

Consumer-based actions to reduce plastic pollution in rivers: A multi-criteria decision analysis approach

Identifying reliable indicators of fitness in polar bears

• How to Write a Great Title
• How to Write an Abstract
• How to Write Your Methods
• How to Report Statistics
• How to Edit Your Work

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The contents of the Writing Center are also available as a live, interactive training session, complete with slides, talking points, and activities. …

There’s a lot to consider when deciding where to submit your work. Learn how to choose a journal that will help your study reach its audience, while reflecting your values as a researcher…

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Reaction vs. Reflection Paper: What’s the Difference?

The purpose of writing a reaction or reflection paper is to provide a deeper and more meaningful understanding of a piece of literature, film, or artwork. As students, it is important to learn how to properly analyze and interpret material in order to gain a better overall understanding and appreciation of it. A reaction paper is a more personal and informal response to the material, while a reflection paper is a more formal and analytical reflection of the material. In this blog post, we will discuss the differences between a reaction paper and a reflection paper, and provide tips for how to write each. We will also discuss the importance of each and how they can be used to gain a better understanding of a given work of art.

ACADS EP.1: Difference between reflection paper and reaction paper

What is a reflection paper?

An essay that focuses on what the author learned from an experience is called a reflection paper. The experience could be completing a task, watching a video, or reading a book. Writers frequently discuss how an experience has altered their perspective on a subject. These essays also contain the authors’ descriptions of the events and their emotions.

What is a reaction paper?

A reaction paper, also known as a response paper, is an essay that expresses the writer’s opinions on a particular subject. These essays typically retain a formal tone while expressing the writer’s opinion and their agreement or disagreement with the concepts presented in a book, article, or film. When writing a reaction paper, authors can also evaluate the piece and provide proof to back up their assertions.

Reaction vs. reflection paper

The following are some key parallels and divergences between a reaction paper and a reflection paper:

Reaction and reflection papers tend to have similar uses. For instance, when they want students to respond to a piece of writing, a book, or a film, teachers and college professors frequently assign them. Students can develop analyses and incorporate evidence when using critical thinking, which is useful practice for writing lab reports and other essays. A reflection paper is typically assigned by a teacher when they want their students to respond to an experience. Students’ communication, teamwork, and organizational skills are frequently enhanced when they share what they learned from a lecture or internship.

Format and tone

Despite the fact that reaction and reflection papers include the writers’ opinions, they typically have a formal tone. These papers maintain their suitability for academic settings thanks to an academic tone and format. Teachers can specify the requirements for their assignments, but most students follow an MLA or AP style manual. Additionally, they employ academic vocabulary and sentence constructions that are less conversational than diary entries. Reaction and reflection papers can range in length and format, but they almost always have an introduction, a body, and a conclusion.

Summary of the work or experience

Writers frequently incorporate a summary of the published work or experience in both types of papers. They try to provide context because they are aware that the reader might not be familiar with their topic. For instance, a writer might briefly summarize a book’s major plot points at the start of their essay. The reader may find it simpler to comprehend the character analyses after reading this explanation. Good reaction and reflection papers frequently include objective summaries that give the reader context without the writer’s personal bias coming through.

The focus of reaction and reflection papers is the primary distinction. Reaction papers highlight the writer’s feelings following a book or video by including their first impressions. Additionally, they are able to evaluate various incidents and offer proof to back up their conclusions. For instance, a student may reference the author’s account of an event to support their claim that they are commenting on a phenomenon that actually exists.

In contrast, a reflection paper focuses more on how the experience or work altered the writer’s perspective. They frequently mention their previous viewpoint and how the subject opened their minds to new concepts. Some reflection papers highlight how the experience or work solidified their preexisting beliefs. For instance, a climate change article could support a student’s conviction that global warming is a real phenomenon. The student may mention this enduring belief in their reflection paper while also highlighting how the assignment exposed them to fresh strategies for tackling global warming.

How to write a reaction or reflection paper

Heres how to write a reaction or reflection paper:

1. Review the reference material

Consider reading the reference material in its entirety if you want to write a strong reaction or reflection paper. You can make sure you comprehend all of the key points by reading the entire book or by watching the entire video. You can also jot down important details to discuss later in notes. As you watch a movie or read a book, for instance, think about writing down any interesting details or queries you have. Try keeping track of your primary responsibilities and interactions with coworkers after each shift if you’re completing an internship.

2. Review your teachers requirements

Before writing your paper, consider reviewing your teachers requirements. You can verify details like word count, formatting type, and whether a reference or works cited page should be included. Knowing the requirements can help you structure your paper and prevent you from having to make revisions later in the writing process.

3. Create an outline

To create an outline, think about using your notes and your teachers’ expectations. Your notes, for instance, could point out three different ways the author introduces a certain theme. These three points can be broken up into paragraphs in your outline, and you can also indicate how long each section should be. Additionally, you can make a note of the quotes and details you want to use in each section.

4. Write an introduction with a thesis statement

The hook in the introduction of reaction and reflection papers entices readers to continue reading. In order to provide context for the reader, it might also include a brief summary of the work or experience. The conclusion of the introduction paragraph should include a thesis statement that sums up your position. If you’re writing a reaction paper, try to summarize your feelings about the work in your thesis statement. These details may also be in the thesis statements for reflection papers, but they usually place more emphasis on how the work or experience shaped your perspective.

5. Write body paragraphs

Writing the body paragraphs that you noted in your outline is the next step. An introduction to the main idea can be made in the topic sentence of each body paragraph. After the topic sentence, go into greater detail about how you analyzed the work or experience and provide evidence to back up your assertions. Although the teacher is typically familiar with the subject you are writing about, you can provide more context if necessary. For example, you can emphasize the main character’s stubbornness if you want to emphasize this quality to make your point.

6. Add a conclusion

Your paper’s conclusion paragraph restates your thesis statement and lists your key points. If you’re writing a reaction paper, you might want to focus on how the piece made you feel or your thoughts on the subject. Reflective essay conclusions could summarize what you learned and how you would persuade others to use your analyses to reevaluate their positions in the future.

Is reaction and reflection the same?

Reaction is largely driven by external stimuli. Contrarily, reflection is a higher-order executive function known as a metacognitive function that calls for awareness and control of one’s own thought process.

What is the difference between summary and reaction paper?

Refine and polish your summary by removing any repetitions or minor details and adding transitions to make the summary read smoothly. The Reaction is a text-based response where you express your opinions regarding the source text.

How do you write a reaction paper?

Write an informative summary of the material. Highlight the work’s main points and important supporting points to condense the content. Use direct quotations from the work to illustrate important ideas. Summarize the content to give the reader a broad understanding of all significant elements of the original work.

What is the purpose of the reaction paper?

In the classroom, reaction papers are frequently used as tools to help students think critically about texts and how they relate to one another or to a larger field of discourse. Research paper topics can also be found in reaction papers.

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In an opinion paper , you will focus on a topic about which you have personal thoughts, beliefs, or feelings.  Your goal is to persuade your reader that your position on this topic is the best one. You won’t accomplish that goal with a rant or diatribe. Instead, you will need to support your claim with facts, statistics, real-life examples or published research studies. So, despite its name, an opinion paper will require some research .

The most common research paper assignment (particularly in undergraduate courses) is a lot like a literature review. You will conduct a thorough search for scholarly sources about your chosen topic, then carefully read and summarize them. But beyond simply describing the books and articles that you read, your goal is to participate in the scholarly “conversation” surrounding your topic. You can do that by:

• Organizing your paper by themes or trends that you discovered in the literature
• Identifying and explaining controversies surrounding your topic
• Pointing out strengths and weaknesses in the studies that you read
• Identifying aspects of the topic that need further research

Sometimes (more commonly in graduate courses), you will design your own study and write about it. While this kind of research paper includes a literature review section, it will also require you to describe your study’s methodology, data analysis and results. The graduate section of Writing@APUS offers advice for students working on original research papers.

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The Effect of Mindfulness on Cognitive Reflection and Reasoning

• ORIGINAL PAPER
• Published: 08 June 2020
• Volume 11 , pages 2150–2160, ( 2020 )

Cite this article

• Stephanie T. Farrar   ORCID: orcid.org/0000-0003-2896-4496 1 ,
• Kielan Yarrow 1 &
• Katy Tapper 1  

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Cognitive decoupling (the ability to distinguish supposition from belief and run thought experiments) is considered one of the key mechanisms in mindfulness, cognitive reflection and reasoning. Therefore, the present study examined whether a brief mindfulness exercise that explicitly encourages cognitive decoupling can increase cognitive reflection and reasoning.

A total of 156 first- and second-year undergraduate students were randomly allocated to either a mindfulness or control condition, before listening to a 15-min audio recording. The mindfulness audio was a recording of the leaves on a stream exercise that focussed on how to dissociate from thoughts (decentring), whereas the control audio was a recording of a book prologue. Cognitive reflection and reasoning were measured through the expanded cognitive reflection test and a syllogistic reasoning test, both of which encourage an incorrect automatic response rather than a correct rational response. The five-facet mindfulness questionnaire-short form and the rational-experiential inventory were also administered as trait measures of mindfulness and thinking style (intuitive or rational), respectively.

The results showed no significant difference between the mindfulness and control conditions on either of the cognitive tests. However, there was a significant positive correlation between trait mindfulness and trait rationality ( r  = 0.56). Further analyses showed that the mindfulness subscales of observing, describing, detaching, and acting mindfully were all significant predictors of trait rationality.

Conclusions

Trait mindfulness and trait rationality are moderately associated, although more research is required to determine whether mindfulness training can increase cognitive reflection and reasoning.

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The data for the study is available from the first author on request.

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STF: designed and executed the study, performed part of the date analysis and wrote the first draft of the manuscript. KY: assisted with the data analysis, wrote part of the results and edited the final manuscript. KT: collaborated with the design of the study and edited the final manuscript. All authors approved the final version of the manuscript for submission.

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Farrar, S.T., Yarrow, K. & Tapper, K. The Effect of Mindfulness on Cognitive Reflection and Reasoning. Mindfulness 11 , 2150–2160 (2020). https://doi.org/10.1007/s12671-020-01429-z

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Difference Between Essay And Reflection Paper

• 1 Introduction
• 2.1 1. Structure
• 2.2 2. Subject matter
• 2.3 3. Purpose
• 2.4 4. Intention
• 2.5 5. Outcome
• 3 Complete your homework by segmenting essays and reflection papers separately.

Introduction

The writing process may differ in various situations and depending on the criteria. Like in childhood, we learned that writing poetry and a story are totally different, and these differences showcase the distinct nature of various papers.

In this article, the discussion will be on essay papers and reflection papers. While many people think that essay papers are almost the same as reflection writing as both share a thinking process in writing. But the distinction is seen in the delivery of these two writing processes.

For instance, in a reflection paper, you will not need to be tidily ordered as it involves both the processes of thinking and learning simultaneously. But, on the other hand, essays have orders and formations as well. So, you need to be in order and then follow your thinking process to prepare an essay.

Where the essay depends on guidelines and structures, you can be a bit frank with reflective writing.

So, the approaches are different, and this is where students find it difficult and confusing to complete their assignments. In addition, when you are on a tight schedule, tension will try to harm your writing process.

Difference between essay and reflection paper

The difference is there, and it will be more clear to you if we tend to state every feature of these two types of the writing process in front of you. Here is what we are exactly going to do.

This article will allow you to understand the crucial difference between these two types of the writing process, and thus, as a student, you will find it easy to complete your homework. Time is money and managing your time is important as a student, especially when you are dealing with your deadline.

Let’s find out the distinctions between essay and reflection papers so that you can avoid confusion and complete your homework on time.

1. Structure

Essays are mostly properly structured and are in order. Tidily ordered essays are mostly considered good essays. Commonly, an essay starts with an introduction, and the majority of it goes to the body part (discussion), and then there is a clear conclusion to state the thinking process.

Moreover, the subject matter needs to be clearly defined in your essay structure.

In contrast, reflective writings are ill-structured, and the subject matter may diffuse. There is no particular structure to reflective writing. In a reflection paper, you start writing, and you continue to do it without following any particular segment.

However, there are various reflective models that you can follow to give a concise shape to your reflective structure.

2. Subject matter

Subject matter in a written paper may seem like you are selecting the topic, and depending on it, you go through the matter. But this is not all.

The subject matter that you will select should not be personal for an essay . The subject matter needs to be clearly defined and specified with the help of proper subject area research.

Moreover, you will need to have a concise idea about which particular subject you are going for and how much information you can gather to complete the writing.

In contrast, a reflection paper and its subject matter can be personal because you will need to experience it before you write it. So, it’s a continuous process that may need more time than an essay to complete.

Moreover, the ideas in reflective paper writing can be drawn from the perspective of the writer. Your forged reflection will be the key to defining the subject matter.

As we have already defined, the purpose of essay writing is mostly pre-determined. When you select a topic, you are likely to know what type of data you are undertaking and how you are going to end it.

So, there is always a concise plan ready to go before you start writing. In this way, the purpose of your writing an essay is also pre-determined, and therefore the reader mostly knows the results.

On the other hand, the only purpose of reflective writing is to deliver a process of learning through thinking and development. This is a continuous development process that considers the gradual delivery of a new understanding of the particular subject matter.

4. Intention

Intentions of writing can change the whole scenario of a paper. For instance, if your intention is to let the audience understand your view on environmental sustainability, then your writing approach and even the subject matter will be based on an experimental process.

On the other hand, if your intention is to be a representative of some process, then the writing approach will be different from the above.

In the case of essay writing, it generally represents the learning of a subject matter. On the other hand, the reflection essay delivers the underlying purpose of the subject matter. And here, you will get to learn new ideas with the development of understanding.

The outcome is set with an essay, whereas the outcome is not confirmed with a reflection paper.

This means that you will get to know what is there in an essay and what it is going to discuss so far in the whole paper.

But reflective papers are not confirmed with their outcomes. This is because it deals with a personal point of view. A writer can forge you to understand their view depending on their perspectives and experience throughout.

Over the course of experience, you will be able to discover new ideas and outcomes in the eyes of a writer.

Complete your homework by segmenting essays and reflection papers separately.

If you are confused, the above-mentioned steps will help you to understand the different writing approaches regarding essays and reflection papers. In this way, you will be able to complete your homework as quickly as possible.

If you do not have much time left to cover both assignments, you can simply ask to do my homework for me . Their expert writers know the distinctions and will deliver you quality writing within the deadline.

Reflection and Refraction of Light: Examples Research Paper

• To find inspiration for your paper and overcome writer’s block
• As a source of information (ensure proper referencing)
• As a template for you assignment

Introduction

Differences between reflection and refraction, reference list.

Light can be regarded as a complex phenomenon due to the fact that it is intangible, and its uses in day-to-day life are innumerable. However, the properties and behaviors of light have been extensively studied by the use of simplified models of light constructed using wave fronts and rays.

A wave front can be defined as a surface that connects light that is produced from a source simultaneously. A ray, on the other hand, is “a thin beam of light that travels in a straight line” (“The Reflection of Light”, 2000, p. 1). By using the stated models, the properties of light can be effectively studied. Some of these properties include the refraction of light and the reflection of light. This paper is an in-depth analysis of the facts surrounding the reflection and refraction of light.

“ Reflection refers to the bouncing of light off a smooth surface.” (Fellers, 2010, p. 1). An example of a common occurrence of light reflection, and that which can easily be seen is the reflection of light off a smooth pool of water. In this case, the environment around the water can be seen in the water because the reflection on the water captures its image.

If the surface f the water is perturbed, waves are formed, which scatter reflected rays, and hence disrupt the reflection (Fellers, 2010). “The angle between the perpendicular line, (the normal), and the incident ray is known as the angle of incidence or the incident angle” (Fellers, 2010, p. 1). On the other hand, “the angle between the normal and the reflected ray is called the angle of reflection” (Fellers, 2010, p. 1).

Laws of reflection

Reflection is governed by one law: “the angle of incidence equals the angle of reflection”. (“The Reflection of Light”, 2000, p. 1). Plane mirrors and other objects whose irregularities are so small that the wavelength of light is larger; light is reflected in only one direction. This phenomenon is termed as specular reflection. However, if a surface is so irregular that light wavelength is smaller than its irregularity, it reflects light in all directions.

This phenomenon is termed as diffuse reflection. Regardless of whether light is reflected in specular reflection or diffuse reflection, the law of reflection is observed for each light ray (on a microscopic scale). There is also another law of reflection that states “the incident ray, the reflected ray and the perpendicular line of impact all fall on the same plane” (Kosman, 2007, p. 1). This is mainly applied in forming geometric optics for curved surfaces like concave and convex mirrors.

Applications of reflection

One of the greatest applications of reflection is the fact that we are able to see objects due to their ability to reflect light off their surfaces. The only exception to this principle is objects that are able to emit light. These are very rare. Reflection is also applied in a number of ways by the use of spherical mirrors. These are used in surveillance, in automobiles, etcetera. Movie screens and projectors are made possible by the phenomenon of reflection. The study of the universe is made possible by the fact that the bodies in our universe reflect some light from the sun (Fellers, 2010).

All of us have at one point experienced refraction of light. When a stick, pencil, straw, and the like are immersed in water, soda, or any other transparent liquid, it is noticeable that the former bends at the exact point where it gets into the liquid. This is an effect of refraction. Refraction can thus be defined as the “bending of light as it enters a denser medium from a less dense medium or as it enters a less dense medium from a denser medium” (Fitzpatrick, 2007, p. 1).

This bending of light is due to the change in the speed at which the light travels in a certain medium due to a higher or lower refractive index. Just like in reflection, the respective angles separated by the normal are called “the angle of incidence and the angle of refraction” (Fitzpatrick, 2007, p. 1).

As light moves to a medium which is less dense, it will be appropriately refracted away from the perpendicular line “The maximum incident angle possible in the denser medium, in order for the refracted ray to just emerge out into the less dense medium is called the critical angle” (Fitzpatrick, 2007, p. 2).

Laws of refraction

The laws of refraction are as follows:

• “The incident ray and the refracted ray lie on either side of the normal to the boundary of separation” (Fitzpatrick, 2007, p. 1).
• “The incident ray, the normal and the reflected ray lie on the same plane, which is called the plane of incidence” (Fitzpatrick, 2007, p. 1).
• “The angle of incidence is greater than the angle of refraction when light enters a denser medium from a less dense medium and vice versa” (Fitzpatrick, 2007, p. 1).
• “The sine of the angle of incidence bears a constant ratio to the sine of the angle of refraction for a given pair. This constant is called the refractive index of the first medium with respect to the second medium” (Fitzpatrick, 2007, p. 1). This is what led t Snell’s law, commonly used in refraction, which is as follows n1sin (angle of incidence) = n2sin (angle of refraction) (Reed, 2009, p. 1)

Applications of refraction

Among the main applications of refraction is the use of refraction in the manufacture of optical lens. These are used for correcting defects in sight like myopia and hyperopia. Refraction is also applied in ophthalmology, in which it is appropriately used to determine if an eye has a refractive error.

In this case, it is also used to determine the best corrective measures for the eye. Refraction is also commonly applied in underwater acoustics and magnifying apparatus like the microscope and the telescope. There is also the common use of the ideas of refraction in the manufacture of Liquid Crystal Display (LCD) screens.

Refraction is also commonly applied in the study of colors. This is enabled by the use of prisms. Refraction is also applied in cameras and magnifying glasses. The twinkling and shimmering of stars is due to the changes in the density of the atmosphere, which make light from the stars to be refracted (Reed, 2009). There is also the phenomenon of mirage which is also an effect of refraction.

Reflection and refraction have a number of differences. One of the main differences between reflection and refraction is the fact that, while refraction can be described as the bending of light, reflection can be described as the bouncing of light. Secondly, in reflection, the incident angle is equal to reflected angle while in refraction; the incident angle is not necessarily equal to the refracted angle.

Most of the applications of reflection are based on the idea of light travelling in air, and being reflected off a surface while most applications of refraction are based on the idea of light travelling within the media refracting the light. Thus in reflection, light travels through air while in refraction, light travels through different media.

Another difference between the two is the fact that reflection is commonly associated with mirrors while its counterpart, refraction, is commonly associated with lens. Another difference between reflection and refraction is the fact that in reflection, light travels in uniform speed, i.e. frequency and wavelength, while in refraction, the frequency and wavelength of light changes, and thus its speed also changes.

Light is a very important part of life. Although sometimes the study of the dynamics of light may seem to be confusing, refraction and reflection aspects of light are quite simple, and they have a myriad of applications. As evidenced in the discussion above, refraction and reflection have a number of differences that distinguish them.

However, these two phenomena of light also have a number of similarities. First of all, they are applicable in all kinds of waves, including light waves. They are also help to make bodies in the universe visible since some bodies reflect light from the sun, while others twinkle due to the difference in the densities of layers of the atmosphere.

Fellers, T., & Davidson, M. (2010). Reflection of Light . Web.

Fitzpatrick, R. (2007). Laws of Refraction at Plane Surfaces, Refractive Index and Critical angle . Web.

Kosman, K. (2007). Laws of Light Reflection. Web.

Reed, R. (2009). Refraction of Light . Web.

Reflection of Light. (2000). Rays and wave fronts . Web.

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IvyPanda . 2018. "Reflection and Refraction of Light: Examples." September 17, 2018. https://ivypanda.com/essays/reflection-and-refraction-of-light/.

1. IvyPanda . "Reflection and Refraction of Light: Examples." September 17, 2018. https://ivypanda.com/essays/reflection-and-refraction-of-light/.

Bibliography

IvyPanda . "Reflection and Refraction of Light: Examples." September 17, 2018. https://ivypanda.com/essays/reflection-and-refraction-of-light/.

Major grocery chain announces first-of-its-kind paper wine bottle: 'A big difference for our planet'

S upermarket chain Aldi has partnered with a sustainable packaging company to create their store-brand paper wine bottles, Packaging Gateway reported. The bottles will be fully recyclable and are part of Aldi's ongoing initiative to reduce the amount of pollution it produces while giving environmentally-conscious shoppers more purchasing options.

While the glass bottles that wine is normally stored in are also recyclable, Aldi's "Frugal Bottles," designed by U.K.-based company Frugalpac, will have several additional environmental benefits. 

For one, the paper-based bottles are five times lighter than glass bottles, making them easier to transport.

The paper bottles will also be made from 94% recycled paperboard and a food-grade pouch.

As is the case with other forms of recycling, though glass is 100% recyclable, far from 100% of it gets recycled. In the United States, somewhere around 33% of used glass is recycled. In the U.K., the number is slightly higher, at 43% . (Some EU countries such as Switzerland and Germany have impressive glass recycling rates of 90%.)

Aldi's decision to come up with a more environmentally friendly wine bottle is reflective of the priorities of the store, as well as of its shoppers.

Watch now: What would Alex Honnold do as dictator for a day?

"Shoppers are striving to become more sustainable in their everyday lives, looking for small ways to make a big difference for our planet," said Aldi UK buying managing director Julie Ashfield.

Other recent Aldi initiatives to improve sustainability include a line of chocolate bars that used responsibly sourced cocoa beans, eliminating plastic bags in its stores, and a monitoring system that allows customers to track plastic they recycle through the store.

It's not just Aldi that is making these types of changes, either. Sustainable packaging is a growing trend, as brands are pressured by the public to reduce the amount of waste they create.

Other recent examples include a British company that created a biodegradable chip bag, a Netherlands-based chain store that uses plant-based packaging , and a new type of packaging made from rice husks .

Join our free newsletter for cool news and actionable info that makes it easy to help yourself while helping the planet.

Major grocery chain announces first-of-its-kind paper wine bottle: 'A big difference for our planet' first appeared on The Cool Down .

The Differences and Similarities between Eukaryotic and Prokaryotic Cells

This essay is about comparing and contrasting eukaryotic and prokaryotic cells. It highlights the structural and functional differences, such as eukaryotic cells having a defined nucleus and membrane-bound organelles, while prokaryotic cells lack these features and have a simpler organization. The essay also discusses similarities, including the presence of a plasma membrane, DNA, and ribosomes in both cell types. It touches on the evolutionary relationship between the two, suggesting that eukaryotic cells evolved from prokaryotic ancestors through endosymbiosis. Understanding these distinctions and similarities is essential for fields like microbiology, genetics, and medicine.

How it works

Eukaryotic and prokaryotic cells epitomize the two cardinal configurations of cellular architecture observed in the biological realm. These cellular variants are delineated by their morphological and functional attributes, reflective of their evolutionary trajectories and ecological roles. Despite their disparities, both eukaryotic and prokaryotic cells partake in vital life processes, rendering them compelling subjects for juxtaposition.

Eukaryotic cells typically manifest greater intricacy compared to prokaryotic cells. They harbor a distinct nucleus ensheathed within a nuclear envelope, housing their genomic material. This compartmentalization affords heightened regulation and oversight over genetic mechanisms.

Moreover, eukaryotic cells harbor a plethora of membrane-bound organelles, including mitochondria, endoplasmic reticulum, Golgi apparatus, and lysosomes. These organelles execute specialized functions that augment the cellular efficacy and intricacy. For instance, mitochondria are lauded as the cellular powerhouses, facilitating adenosine triphosphate (ATP) synthesis through cellular respiration. Conversely, the endoplasmic reticulum and Golgi apparatus are implicated in protein and lipid biosynthesis and conveyance.

Prokaryotic cells, exemplified by bacteria and archaea, lack a well-defined nucleus and membrane-bound organelles. Their genomic material resides in a nucleoid region devoid of a membrane enclosure. This rudimentary organizational schema bespeaks their primordial evolutionary lineage and facilitates rapid proliferation and reproduction. Prokaryotes typically exhibit diminutive proportions compared to eukaryotes, augmenting their favorable surface-area-to-volume ratio. This ratio bestows advantages in nutrient assimilation and waste expulsion. Additionally, prokaryotic cells often possess cell walls comprised of peptidoglycan, providing structural reinforcement and protection. Certain prokaryotes also feature external appendages such as flagella and pili, which facilitate motility and adherence to substrates.

One of the most conspicuous disparities between eukaryotic and prokaryotic cells pertains to their dimensions. Eukaryotic cells typically exhibit substantially larger dimensions, ranging from 10 to 100 micrometers in diameter, whereas prokaryotic cells typically measure between 0.1 and 5 micrometers. This pronounced size differential exerts profound influences on cellular intricacy and functionality. Larger eukaryotic cells can accommodate a myriad of organelles and internal structures, fostering intricate life processes. Conversely, the diminutive dimensions of prokaryotic cells expedite rapid molecular diffusion across the cellular membrane, bolstering their rapid growth rates and adaptability.

Despite these disparities, eukaryotic and prokaryotic cells share several fundamental attributes. Both cell types are ensconced by a plasma membrane, which orchestrates the ingress and egress of substances. Moreover, they both utilize deoxyribonucleic acid (DNA) as their hereditary blueprint and rely on ribosomes for protein synthesis. Foundational metabolic pathways such as glycolysis and the citric acid cycle are conserved across both cell types, underscoring their shared evolutionary lineage. Additionally, both eukaryotic and prokaryotic cells exhibit a panoply of forms and functions, adapting to diverse environmental exigencies.

The evolutionary nexus between eukaryotic and prokaryotic cells presents another intriguing facet. It is widely posited that eukaryotic cells originated from prokaryotic antecedents via endosymbiosis. As per this hypothesis, select prokaryotic cells were engulfed by larger host cells, eventually evolving into organelles such as mitochondria and chloroplasts. This symbiotic alliance conferred a discernible selective advantage, catalyzing the evolution of intricate eukaryotic cells. Substantiating evidence includes the presence of double membranes surrounding these organelles and their circular DNA akin to bacterial genomes.

Comprehending the dichotomies and parallels between eukaryotic and prokaryotic cells is indispensable across diverse scientific domains, encompassing microbiology, genetics, and evolutionary biology. These insights enrich our understanding of cellular functionalities, the origins of life, and the evolution of complex organisms. Moreover, this comprehension engenders practical ramifications in medicine and biotechnology. For instance, discerning the distinctions between prokaryotic and eukaryotic cells is pivotal in devising antibiotics efficacious against bacteria whilst sparing human cells.

In conclusion, while eukaryotic and prokaryotic cells diverge significantly in their morphological, functional, and dimensional attributes, they converge on fundamental biological processes and evolutionary origins. Eukaryotic cells are characterized by their compartmentalized nucleus and organelles, whereas prokaryotic cells exhibit a simpler, archaic morphology. Notwithstanding these disparities, both cellular variants execute indispensable life functions and contribute to terrestrial biodiversity. Recognizing these congruities and divergences amplifies our comprehension of biology and informs a spectrum of scientific and medical breakthroughs.

Remember, this essay serves as a springboard for inspiration and further inquiry. For bespoke guidance and assurance of academic rigor, consider enlisting the expertise of professionals at EduBirdie.

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• Open access
• Published: 24 May 2024

For the rural curious: mixed methods evaluation of a rural pharmacy practice elective

• Timothy P. Stratton 1  

BMC Medical Education volume  24 , Article number:  573 ( 2024 ) Cite this article

134 Accesses

Metrics details

As of 2020, 20% of people residing in the United States of America (U.S.) lived in rural communities. Despite rural residents tending to be older, poorer, and having greater disease burden than their urban counterparts, the number of rural primary care providers continues to decline. Nearly 66% of U.S. Primary Care Health Professional Shortage Areas are designated as rural. Pharmacists can help address this shortage of rural primary care providers, often serving as providers of first-contact care; however, only 12% of U.S. pharmacists practice in rural communities. To help address this gap, in 2022 an elective Rural Pharmacy course was created at the University of Minnesota College of Pharmacy by a faculty member who has rural practice experience.

The course combines formal lectures, guest presentations by rural pharmacists and student interviews with additional rural pharmacists. For the 42 students enrolled in the course in 2022 and 2023, non-parametric statistics were used to compare the percentage of students who were raised in rural communities or who otherwise had extensive exposure to rural, and compare student interest ratings (1 to 7) about practicing/living rural at the beginning and end of the course. Students also wrote end-of-course reflection papers, commenting on the course and their interviews with rural pharmacists.

Across both years, 45% of the enrolled students had previous experience in rural communities. The net change in Rural Interest scores among students completing both questionnaires was + 5 in 2022 and + 2 in 2023, both non-significant differences. The largest shifts in student interest were from “Not Sure” at the start of the course to “Interested” or “Not Interested” at the end of the course, and from “Interested” to “Very Interested.” In their reflection papers nearly 60% of students reported being most impressed by their interviews with rural pharmacists.

Conclusions

A course addressing the benefits and challenges of practicing pharmacy in rural communities was well-received by pharmacy students. Even students who have little interest in living in a rural community can benefit from being introduced to rural culture, enabling them to provide more culturally-responsive care for patients from rural communities.

Peer Review reports

Introduction

The Unites States (U.S.) Census Bureau redefined “rural” for the 2020 census as communities with populations of fewer than 5,000 people (fewer than 2,000 housing units) and located more than 1.5 miles (2.4 km) from a high-density urban area. Based on this revised definition, 20% of the U.S. population in 2020 lived in rural communities [ 1 ]. The percentage of rural residents varied greatly by region, with only 11% of people in the West Region residing in rural areas, followed by 16% in the Northeast Region, 24% in the South Region and 26% in the Midwest Region [ 2 ]. At the extremes, fewer than 6% of people in California lived in rural areas, while nearly 65% of Vermont residents lived in rural areas [ 1 ]. In contrast, as of 2021 the U.S. Bureau of Labor Statistics reported that only 12% of the nation’s pharmacists practiced in nonmetro (rural) communities [ 3 ].

On average, rural residents tend to be older [ 4 ], poorer [ 5 ], experience greater disease burden [ 6 ] and lack health insurance or be underinsured [ 7 ] than residents of urban communities. The average age and disease burden among rural residents is increasing due to outmigration of young adults from rural to urban communities and the in-migration of older adults to rural communities following retirement [ 4 ]. Yet as the proportion of older residents in rural communities continues to increase, the availability of primary care providers in rural communities continues to decrease. Nearly 66% of Primary Care Health Professional Shortage Areas in 2023 are designated as rural [ 8 ]. Pharmacists can be part of the solution to address the existing and anticipated shortage of primary care providers in rural communities [ 9 ]. Rural pharmacists often serve as “providers of first-contact care” for patients who are seeking to self-treat a health condition [ 10 ]. Where self-treatment is inappropriate, the pharmacist is in a position to refer the patient to appropriate professional care.

This paper describes a new course taught in the Doctor of Pharmacy (PharmD) program in the University of Minnesota College of Pharmacy that introduces students to the unique benefits and challenges of practicing pharmacy in rural communities.

One college, two campuses

The University of Minnesota (UMN) is a public, research-intensive (Carnegie R1) institution. The UMN College of Pharmacy opened on the Minneapolis campus in 1892 [ 11 ]. Prior to 2003, the College of Pharmacy included four departments: Experimental & Clinical Pharmacology, Medicinal Chemistry, Pharmaceutics, and Pharmacy Care & Health Systems. However, to address a shortage of pharmacists in Greater Minnesota – counties outside of the seven-county Minneapolis-St. Paul Twin Cities Metro Area [ 12 ] – in 2003 the College of Pharmacy expanded its program 150 miles (241 km) north to Duluth on the University of Minnesota Duluth campus, adding a fifth department to the College, Pharmacy Practice and Pharmaceutical Sciences (PPPS).

The specific multi-campus model used by the UMN College of Pharmacy is somewhat unique among multi-campus pharmacy programs in the U.S. The PPPS department includes faculty representing Biochemistry and each of the major Pharmacy disciplines (Clinical Pharmacy, Medicinal Chemistry, Pharmaceutics, Pharmacology, Pharmacy Practice, and Social & Administrative Pharmacy). Didactic courses within the College are taught using videoconferencing technology, with classroom presentations/lectures originating from either Minneapolis or Duluth and being broadcast to the other campus.

The mission of the PPPS department includes preparing pharmacists to provide patient care in rural and Indigenous communities [ 13 ]. PPPS faculty embody this mission in all four areas of an academic health professions program, highlighting the unique health needs of rural residents in their teaching, addressing these needs through community-based participatory research [ 14 ], conducting service activities in rural communities, and providing clinical services. Until 2022, however, no single course in the College of Pharmacy’s curriculum was devoted specifically to rural health.

Rural pharmacy elective-course description and structure

To help address this gap in the College of Pharmacy curriculum, the author – a pharmacist who has practiced hospital, community and long-term care pharmacy in frontier/Indigenous communities in Alaska [ 10 ], Eastern Montana and Minnesota – created a two-credit elective course (two hours per week for 15 weeks) in Rural Pharmacy to introduce students to the benefits and challenges of living and practicing in rural communities. Development of the course was guided by the author’s teaching philosophy; to paraphrase Confucian philosopher Xun Kuang [ 15 ]: “Tell me and I will forget. Show me and I will remember. Involve me and I will understand.”

The Rural Pharmacy course was designed as a HyFlex course [ 16 ] that allows the learner to choose by which content delivery method they would like to learn. Learners in a Hyflex course may elect to attend a live class session in person in a classroom, may attend a live class session remotely via videoconference, or may learn online anytime. Each live class session is recorded to accommodate students who prefer to learn online during a given week, or throughout the entire course.

The Rural Pharmacy elective is a “modified” HyFlex design in that no in-person option is available. University of Minnesota College of Pharmacy faculty and students are accustomed to videoconferencing as a course delivery method, the college having used videoconference technology since 2003 to conduct live, in-person sessions for learners on campuses located 2.5 h apart from one other. Required and elective didactic courses delivered by videoconference are always recorded, enabling learners to view the recording at a more convenient time if they are unable to attend the live class session. Another reason that an in-person option for the Rural Pharmacy elective is not offered is that live course sessions are conducted in the evening to accommodate students from different years in the pharmacy program (P2 and P3) whose other courses are all on different schedules, and Minnesota’s frequent snowy and icy winter conditions are not always conducive to safe travel to and from campus, especially at night.

At the time of this writing, during the first three pre-clinical years University of Minnesota College of Pharmacy students are required to complete 15 credits of elective courses above and beyond their required courses. The Rural Pharmacy elective is open to students in the final two pre-clinical years of the PharmD program (P2 and P3), but enrollment is capped by the instructor at 25 students per offering. Live class sessions are conducted once weekly for two hours in the early evening by videoconference for all students, whether based in Duluth or in the Minneapolis-St. Paul Twin Cities area. The early evening hours avoid conflicts with students’ other courses, which are on different schedules between 8:00 am and 5:30 pm for both of the two years. Students are encouraged to attend as many live videoconference sessions as possible, especially when a guest presenter is scheduled; however, as noted above all class sessions are recorded for viewing or reviewing at a more convenient time. The recordings accommodate students who may be working in a pharmacy as a Pharmacy Intern or Pharmacy Technician at the time class is scheduled, or students who desire to review one or more recorded class sessions prior to the written midterm examination.

A University of Minnesota Post-Graduate Year 1 (PGY1) Rural Pharmacy Resident [ 17 ] serves as the Teaching Assistant for the course each year, participating in the live class sessions via videoconference. The Pharmacy Resident is based out of a rural community in central Minnesota, traveling to two other rural communities and providing comprehensive medication management services [ 18 ] to residents of all three communities. While maintaining patient confidentiality, the Resident shares with students their experiences caring for patients in rural communities, some stories being only a few hours old. In addition to regularly participating in live class sessions, the TA prepares and leads a class session on their own, and conducts the live session interviews with guest rural pharmacists as described below.

About half of the class sessions feature guest pharmacists who currently practice in rural communities, guests joining the live class sessions via videoconference. When a guest pharmacist is invited to participate in the course, the instructor provides the pharmacist with a list of potential interview questions that they would be asked to address during the class session. On rare occasions the visits with pharmacist(s) are pre-recorded either to better accommodate the pharmacist’s work schedule or because of time zone differences between Minnesota and the states where the pharmacists live/work. Pre-recorded interviews are played during the live class session, and students submit questions they would have asked the pharmacist had the pharmacist been able to join the class session in real time. Those questions are then summarized by the instructor and forwarded to the guest pharmacist to respond to as the pharmacist’s time allows. Pharmacists living and practicing in rural and Indigenous communities from throughout Minnesota and from as far away as Alaska have participated in the live sessions, either pre-recorded or in real time. In addition to rural pharmacists, guest presenters have included Advance Practice Nurses [ 19 ] from rural communities, and a Biologist who works with an Indigenous community on the impacts of climate change on the health of the community.

A variety of assessments are utilized in the course including reflection papers, an online multiple-choice/true–false/short answer midterm exam, written participation in online discussions, in-class student presentations and written summaries of interviews with pharmacists practicing in rural communities. The course is graded on a A,B,C,D,F letter grade scale. A total of 300 points are available across nine activities in the course, ranging in value from 5–50 points. The grading scale used in the course is the professional scale used in all of the college’s courses, an A grade being attained by students who earn at least 93% of the available points while students earning fewer than 60% of available points do not receive a passing grade. The possible number of points available on individual assignments are assigned by the instructor based on the amount of time and effort students are expected to expend on the assignment as well as the quality of each assignment’s deliverable.

At the start of the course students complete a brief 7-point Likert-type questionnaire regarding their familiarity with rural communities and interest in possibly practicing in a rural community. The questionnaires are confidential rather than anonymous as students complete the same questionnaire again at the end of the course. The course director uses student names to match start-of-course and end-of-course questionnaires to measure changes in student attitudes. Students also write a brief paper describing their experiences with rural communities and the reason for their interest in learning (or learning more) about living and practicing in rural communities. The instructor uses this information to tailor presentations in the course for the entire class based on the students’ familiarity with rural communities. This information also familiarizes the instructor with students’ backgrounds, enabling the instructor to invite specific students to share their rural experiences as relevant opportunities arise during live class sessions. The initial questionnaire and interest paper collectively constitute 8.37% of the course grade.

The online midterm examination is based on material provided in the textbook [ 20 ] or during instructor or Resident presentations. Students are tested on their knowledge about what constitutes “rural” as defined by several different U.S. government agencies, rural culture, challenges in rural public health, and opportunities and challenges related to practicing pharmacy in rural communities. The midterm exam score constitutes 16.7% of the course grade.

As mentioned previously, the HyFlex nature of the course accommodates students who are unable to attend the live videoconference sessions. All students, however, participate in weekly written online discussions based on the live videoconference session from that week. Live sessions are recorded so that any student may view and listen to the session at their leisure. In the online discussion, students are asked to respond to an instructor-generated question based on that week’s live class session. Students are asked to post their response first, then comment on the response of at least one other classmate. The Canvas learning management system [ 21 ] facilitates this learning approach, providing the instructor the option to require a student to post their response before reading the responses of classmates. Students who post their responses by the weekly deadline receive full participation credit for the week, rather than being graded on the length of their response or on the number of responses they make to classmates’ postings. As a HyFlex course, students are not awarded extra points for attending the live videoconference session, nor are they penalized for not participating in the live videoconference session. Participation constitutes 16.7% of the course grade.

Indigenous people began living in what today is referred to as Minnesota some 13,000 years ago. Among the earliest identifiable tribes in Minnesota were the Dakota (Sioux) circa 1000 CE and the Anishinaabe (Chippewa, Ojibwe) who arrived in the mid-1700s [ 22 ]. Today, Minnesota is home to four Dakota and seven Anishibaabe reservations [ 23 ], most of these communities being located in rural or frontier Minnesota counties. In contrast to these early inhabitants whose ancestors have lived in Minnesota for hundreds of years, today foreign immigrants are arriving in Minnesota in increasing numbers [ 24 ]. Many of these new arrivals settle in communities outside of the Twin Cities Metro Area [ 25 ]. This spectrum of diversity underlies the importance for healthcare providers to learn to provide culturally-responsive care [ 26 ]; therefore, students in the course learn about Indigenous people or foreign-born immigrants they might encounter if practicing in rural Minnesota. Each student is assigned a particular culture (not their own), and through readings about and/or interviews with members from that culture prepares a brief presentation they share with the class during a live videoconference session. Again, because this is a HyFlex course a student who knows in advance that they will be unable to attend class when they are scheduled to present are able to pre-record their presentation. Pre-recorded student presentations are played during the live course session. This exercise constitutes 16.7% of the course grade.

As students in this course are training to become pharmacists, they interview pharmacists who currently practice in rural communities (or who have practiced in a rural community in the recent past). These interviews supplement the rural pharmacy practice stories provided by the instructor, the Resident, and the pharmacists who present during class videoconference sessions. Most, but not all, of the pharmacists who participate in the course are the instructor’s former students from the UMN College of Pharmacy, Duluth. In addition to pharmacists with practice experience in rural Minnesota, pharmacists in the instructor’s circle of contacts from rural Alaska, Wisconsin and Michigan have participated in the course, as have pharmacists from four different rural Indian Health Service [ 27 ] /Tribal Health Clinics. Potential pharmacist participants are contacted by the course instructor before the course begins to gauge their interest and willingness to participate in a live class session or be interviewed by the students, and are provided with the list of interview questions that will be asked. Characteristics and practice settings of the pharmacists who participated during the first two offerings of the course are presented in Table  1 .

The instructor assigns the students to interview teams of two to three students who conduct structured interviews with the rural pharmacists who practice in community, critical access hospital [ 28 ], health system hospital or Indian Health Service/Tribal Health settings. Each student is assigned to one team to interview a community pharmacist, and then to a different team to interview the health system pharmacist. Where possible, teams are structured to reflect gender diversity and include students from different years in the pharmacy program. Each student team contacts their assigned pharmacist and schedules a telephone or videoconference interview. Interviews are intended to last no more than 30 min, but oftentimes go longer as the conversations between the students and the pharmacist range far beyond the structured questions provided by the instructor.

Each student submits written summaries of their two interviews. Each interview team provides informal presentations about their interviews to the class during a live videoconference class session. Each interview assignment constitutes 16.7% of the course grade.

At the end of the course, students once again complete the 7-point Likert-type questionnaire regarding their interest in possibly practicing in a rural community. The numerical results from this questionnaire are compared to the numerical results of the interest questionnaire that the student completed at the start of the course. Each student also writes a brief reflection paper regarding what they learned in the course about practicing pharmacy in a rural community, and what aspect of the course they found most interesting/helpful in their learning. As with the similar assignments at the beginning of the course, the final questionnaire and final reflection paper constitutes 8.37% of the course grade.

Rural pharmacy elective-topics

Topics presented in the course are listed in Table  2 . Topics for didactic sessions early in the course are based on selected chapters from the textbook required for the course, Foundations of Rural Public Health in America (2022), by Joseph N. Inungu and Mark J. Minelli [ 20 ]. The course also features interdisciplinary and interprofessional components. As noted earlier, one guest presenter is a PhD Biologist employed by one of Minnesota’s American Indian tribes. That individual addresses Climate Justice, explaining the impact of climate change on rural Indigenous communities. Also as noted earlier, a group of rural Advanced Practice Nurses in different subspecialties present a panel session addressing the challenges faced by the communities they serve, and describe how they interact with rural pharmacists in their communities.

Assessing course outcomes

The percentages of students enrolled in the course on each campus who reported growing up in a rural community or having spent considerable time visiting relatives who lived in rural communities were compared using Fisher’s exact test [ 29 ]. For students completing rural interest questionnaires both at the beginning and the end of the course, rating scores from both years and both campuses were combined and paired. Given the ordinal nature of the data, beginning/end of course ratings were evaluated using the Wilcoxon signed-rank test [ 30 ]. A two-tailed alpha value of 0.05 was selected as the criterion to indicate significance in all numerical comparisons.

For the first offering of the course in Spring, 2022 a total of 25 students completed the course. Spring 2023 had 17 students in the course. The demographics of the students in these two cohorts are summarized in Table  3 .

Between the first two offerings of this course, 25 students on the Minneapolis campus enrolled in the course. Of these 25, 10 (40%) reported growing up in a rural community or having spent considerable time visiting relatives who lived in rural communities. Among the 17 Duluth students enrolled in the course between the two years, nine (53%) reported having grown up or otherwise spent considerable time in rural areas. This difference was not statistically significant.

At the beginning and end of the course, students rated their interest in living/practicing in rural community using a 7-point Likert-type scale ranging from “1-No interest” to “7-When can I start?!” The results from the 36 students who completed both the pre and post questionnaires are presented in Fig.  1 .

Interest in Practicing Pharmacy in Rural Communities ( n  = 36)

The total net change in Rural Interest scores across all students completing both questionnaires was + 5 in 2022 and + 2 in 2023, some student scores increasing, others decreasing, and still others remaining the same. Results of the Wilcoxon Signed-Ranks Test were non-significant ( z  = -1.5903; p  = 0.112).

The largest change in scores occurred in the “Not sure” category (middle choice), with only one student remaining unsure of their interest in practicing in a rural community at the end of the course compared to six students at the beginning of the course. Four students who selected “Not sure” at the start of the course expressed lower interest in practicing in a rural community at the end of the course, one of these students moving down three levels from “Not sure” to “No interest.” One student who had selected “Interested” at the beginning of the course also dropped three levels at the end of the course to “Slight interest.” In contrast, several students who had selected “Interested” at the start of the course moved up to “Very Interested” or “When can I start?!”.

At the end of the course, students were asked to reflect on the impact of the course on their interest in practicing pharmacy in a rural community. Among the 42 students enrolled in the course during the first two years, 25 students in their reflection papers explicitly expressed appreciation for being able to interview pharmacists currently practicing in rural communities, while 20 explicitly expressed appreciation for having rural pharmacists and other professionals as guest speakers during class sessions. Two word clouds were generated from students’ reflection papers, one based on student perceptions of the benefits of living/practicing in a rural community (Fig.  2 ), and the other based on student perceptions of the challenges of living/practicing in a rural community (Fig.  3 ).

Word cloud featuring perceived benefits of living and practicing pharmacy mentioned in Rural Pharmacy students’ end-of-course reflection papers. “Courtesy of FreeWordCloudGenerator.com”

Word cloud featuring perceived challenges of living and practicing pharmacy mentioned in Rural Pharmacy students’ end-of-course reflection papers. “Courtesy of FreeWordCloudGenerator.com”

Representative student comments excerpted from their reflection papers regarding what they had heard from rural pharmacists who participated in the course are provided below. Each student’s comment is followed by that student’s final rating of their interest in practicing pharmacy in a rural community (1 = No interest, 7 = When can I start?!):

Before this course I had no interest in practicing rural before but now I’d at least entertain the idea after speaking and interviewing pharmacists that did or currently practice there. (Student selected ratings of 1 and 2) Hearing so many amazing stories, pharmacists are truly more than just “pill counting” because a single pharmacy can connect them with other rural health professionals, expanding the capabilities of rural pharmacists…. (2) If you can dream it you can do it in rural pharmacy. (5) It was great to have [the pharmacist I interviewed] in my network, as [they] said I can contact [them] anytime with questions outside… [of] my interview. I learned that having many contacts in your network, especially in rural areas, is so important…. (6) This class stimulated a future career interest that I already had, but was not sure exactly how to get started and who to ask if I had any questions. I feel like I now have many resources to reach out to when it comes to my future career, which makes me incredibly happy and comfortable. (7)

Students also expressed appreciation for other aspects of the course, whether the students were interested in practicing in a rural community at the end of the course or not. Again, each student’s comment is followed by that student’s final rating of their interest in practicing pharmacy in a rural community (1 = No interest, 7 = When can I start?!):

Even if I do not practice as a rural pharmacist, I will value the exposure and learning that has come from the topics covered in this course. (3) To be frank, I never even entertained the idea of practicing as a rural pharmacist. I’ve always wanted to work in an urban ambulatory care setting…. I did not expect the class to be as eye opening as it truly was…. I’m much more open to serving in a rural community and may consider it strongly . (3) It would be a huge adjustment to move to a rural area since I have grown up in [an urban community] my whole life. I want to work in a rural community since it is rewarding, but it is difficult to leave family behind and essentially start a new life with new people. (4) This is a rural pharmacy class, but it did not feel biased towards only working rural…. I came into this class knowing that I had an interest in rural pharmacy, but I did not expect to come out of this class even more interested in what rural areas have to offer. (6) Before starting this course, I knew that I wanted to practice pharmacy in a rural community…. Many times during this course we stated, “When you’ve seen one rural community, you’ve seen one rural community.” I did not know how true this statement was before this course…. Despite their vast differences, one common underlying theme is the health disparities seen in rural areas. (7)

It is important that health professions students be introduced to rural culture, even if they are “never” going to live/practice in a rural community themselves. With 5–64% of states’ populations living in rural communities [ 1 ], the odds are good that at some point in their careers, health professionals living in large urban centers are going to care for patients who have come from rural communities to receive more specialized care than is available locally [ 31 ]. Being introduced to rural culture can help students provide more culturally responsive care [ 32 ] to patients from rural communities during their careers.

The purpose of this course was to introduce pharmacy students to the advantages and challenges of practicing and living in rural communities. The course was not intended to “change hearts and minds” of students regarding their possible interest in practicing in a rural setting, and as can be seen from the results, students’ “interest in rural” ratings collectively neither significantly increased nor decreased between the beginning and the end of the course. Regardless, from comments in their reflection papers students generally appreciated the course, finding the interviews with rural pharmacists to be particularly valuable. This finding was heartening to the instructor who was initially concerned about the amount of out-of-class work being asked of the students.

Likewise, guest presenters who participated in the live class sessions and pharmacists interviewed by the students informally expressed their satisfaction with participating in the course, and expressed gratitude that this course was being offered. One pharmacist who previously practiced in a remote Alaska community but had recently moved to a major urban center in the “Lower 48” (Alaskan reference to states in the contiguous United States south of the 49th Parallel) expressed how much they enjoyed sharing their stories with the Rural Pharmacy students. The students with which this pharmacist currently works all desire to practice in large urban centers and are not particularly interested in hearing about the pharmacist’s experiences practicing in small, isolated communities. Another pharmacist noted that they really appreciated joining the students virtually in the live classroom, and was going to recommend this approach to other pharmacy schools with which they work as a way to generate interest in rural pharmacy in general, as well as interest in their particular pharmacy as a clinical rotation site.

A few changes were made in the roster of pharmacists participating in the course from year to year; however, most of the guest speakers and pharmacists who were interviewed by the students participated in the course both years. Another change being considered for the next offering of the course is to add a live videoconference session with a Minnesota Department of Agriculture “Farm Counselor” (a Licensed Professional Counselor) who makes in-person “farm calls” to address farm families’ mental health needs within the unique context of farm culture [ 33 ] (MN Dept of Ag, 2023).

A course specifically addressing the benefits and challenges of practicing pharmacy in rural communities was well-received by pharmacy students enrolled in the course, and by the rural guest presenters and rural pharmacists who were interviewed by the students. Even students who have little interest in living or practicing in a rural community can benefit from being introduced to rural culture, helping all students provide more culturally-responsive care for patients from rural communities.

Availability of data and materials

The data analyzed during the current study are not publicly available due to stipulations in the U.S. Family Educational Rights and Privacy Act (FERPA), but are available in de-identified form from the corresponding author on reasonable request.

Abbreviations

Students enrolled in years 1, 2 or 3 of the Doctor of Pharmacy (PharmD) program

Post-Graduate Year 1

Doctor of Pharmacy

Doctor of Philosophy

Pharmacy Practice and Pharmaceutical Sciences

Teaching Assistant

University of Minnesota

United States of America

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TPS is Professor of Pharmacy Practice in the University of Minnesota College of Pharmacy, Duluth. He has practiced community, hospital and long-term care pharmacy in frontier communities in Southeast Alaska, and at Indian Health Service/Tribal Health clinics in frontier Alaska and eastern Montana, and in rural Minnesota. He is a member of the Rural Pharmacy Consortium , a Past Chair of the Small and Rural Hospital Section Advisory Group for the American Society of Health-System Pharmacists, and a Past President of the Minnesota Rural Health Association.

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Stratton, T.P. For the rural curious: mixed methods evaluation of a rural pharmacy practice elective. BMC Med Educ 24 , 573 (2024). https://doi.org/10.1186/s12909-024-05539-3

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The gut microbiome (GM) modulates body weight/composition and gastrointestinal functioning; therefore, approaches targeting resident gut microbes have attracted considerable interest. Intermittent fasting (IF) and protein pacing (P) regimens are effective in facilitating weight loss (WL) and enhancing body composition. However, the interrelationships between IF- and P-induced WL and the GM are unknown. The current randomized controlled study describes distinct fecal microbial and plasma metabolomic signatures between combined IF-P ( n  = 21) versus a heart-healthy, calorie-restricted (CR, n  = 20) diet matched for overall energy intake in free-living human participants (women = 27; men = 14) with overweight/obesity for 8 weeks. Gut symptomatology improves and abundance of Christensenellaceae microbes and circulating cytokines and amino acid metabolites favoring fat oxidation increase with IF-P (p < 0.05), whereas metabolites associated with a longevity-related metabolic pathway increase with CR (p < 0.05). Differences indicate GM and metabolomic factors play a role in WL maintenance and body composition. This novel work provides insight into the GM and metabolomic profile of participants following an IF-P or CR diet and highlights important differences in microbial assembly associated with WL and body composition responsiveness. These data may inform future GM-focused precision nutrition recommendations using larger sample sizes of longer duration. Trial registration, March 6, 2020 (ClinicalTrials.gov as NCT04327141), based on a previous randomized intervention trial.

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Introduction.

As a principal modulator of the gut microbiome (GM) and weight status, nutritional input holds great therapeutic promise for addressing a wide range of metabolic dysregulation 1 . Dependent on the host for nutrients and fluid, one of the main processes by which the GM affects host physiology is producing bioactive metabolites from the gastrointestinal (GI) contents. Nutrient composition, feeding frequency, and meal timing impact this dependency 2 , 3 . To maintain a stable community and ecosystem, the GM must regulate its growth rate and diversity in response to nutrient availability and population density 4 . Such maintenance is affected by caloric restriction (CR) coupled with periods of feeding and intermittent fasting (IF) 5 . Moreover, we’ve recently shown the nutritional composition and meal frequency during these periods alter the metabolizable energy for the host 6 . The current study incorporates protein pacing (P), defined as four meals/day consumed evenly spaced every 4 h, consisting of 25–50 g of protein/meal 7 , 8 , 9 . Indeed, we have previously characterized a dietary approach of calorie-restricted IF-P combined and P alone 7 , 8 . These studies included nutrient-dense meal replacement shakes, along with whole foods, to quantitatively examine beneficial changes in body composition and cardiometabolic, inflammatory, and toxin-related outcomes in healthy and overweight individuals 7 , 8 , 10 , 11 , 12 . Further, recent preclinical work in mice has identified dietary protein as having anti-obesity effects after CR that are partially modulated through the GM 13 . Thus, the need to examine this in humans is warranted.

In this current work, we compare the effects of two low-calorie dietary interventions matched for weekly energy intake and expenditure; continuous caloric restriction on a heart-healthy diet (CR) aligned with current United States (US) dietary recommendations 14 versus our calorie-restricted IF-P diet 8 , 15 , in forty-one individuals with overweight or obesity, over an 8-week intervention. We hypothesize an IF-P diet may favorably influence the GM and metabolome to a greater extent than a calorie-matched CR alone. This exploratory investigation utilizes data and samples from a randomized controlled trial (NCT04327141) that compares the effects of the CR versus IF-P diet on anthropometric and cardiometabolic outcomes, as previously published 15 . As an additional analysis, we select “high” and “low” responders based on relative weight loss (WL) for a subgroup examination of the IF-P diet to better elucidate potential differential responses to intermittent fasting and protein pacing. Of special note, one individual lost 15% of their initial body weight over the 8-week intervention; this individual is followed longitudinally for a year to explore the dynamics of their GM and fecal metabolome. Novel findings from the current study shows an IF-P regimen results in improved gut symptomatology, a more pronounced community shift, and greater divergence of the gut microbiome, including microbial families and genera, such as Christensenellaceae , Rikenellaceae , and Marvinbryantia , associated with favorable metabolic profiles, compared to CR. Furthermore, IF-P significantly increases cytokines linked to lipolysis, weight loss, inflammation, and immune response. These findings shed light on the differential effects of IF-P as a promising dietary intervention for obesity management and microbiotic and metabolic health.

Intermittent fasting - protein pacing (IF-P) significantly influences gut microbiome (GM) dynamics compared to calorie restriction (CR)

We compared an IF-P vs. a CR per-protocol dietary intervention (matched for total energy intake and expenditure) over eight weeks to compare changes in weight, cardiometabolic outcomes, and the GM in men and women with overweight/obesity (IF-P: n  = 21; CR: n  = 20). One participant in each group were lost to follow-up due to non-compliance with dietary intervention (Fig.  1a ; CONSORT flow diagram: Supplementary Fig.  S1a ). The primary outcomes of dietary intake, body weight and composition responses, cardiometabolic outcomes, and hunger ratings after both dietary interventions are provided in our companion paper 15 . Briefly, after a one-week run-in period consuming their usual dietary intake (baseline diet), with no differences between groups at baseline for any dietary intake variable 15 , both dietary interventions significantly reduced total fat, carbohydrate, sodium, sugar, and energy intake by approximately 40% (~1000 kcals/day) from baseline levels (Fig.  1b ; Supplementary Data  1 ). By design, IF-P increased protein intake greater than CR during the intervention. The IF-P regimen consisted of 35% carbohydrate, 30% fat, and 35% protein for five to six days per week and a weekly extended modified fasting period (36–60 h) consisting of 350–550 kcals per day using randomization, as detailed previously 7 , 8 , 9 , 10 , 15 . In comparison, the CR regimen consisted of 41% carbohydrate, 38% fat, and 21% protein in accordance with current US dietary recommendations (Supplementary Table  S1 ) 14 , 16 . Using two-way factorial mixed model analysis of variance (ANOVA), significant macronutrient decreases drove energy reduction from dietary fat and carbohydrate ( p  < 0.001), with increased protein in the IF-P compared to CR ( p  < 0.001; Supplementary Fig.  S1b ; Supplementary Data  1 ). Regarding GI functioning and GM modulation, IF-P significantly decreased sugar and increased dietary fiber relative to CR (IF-P; pre, 20 ± 2 vs. post, 26 ± 2: CR; pre, 24 ± 3 vs. 24 ± 2 g/day; p  < 0.05). Despite similar average weekly energy intake (~9000 kcals/week) and physical activity energy expenditure (~350 kcals/day; p  = 0.260) during the intervention, participants following the IF-P regimen lost significantly more body weight (−8.81 ± 0.71% vs. −5.40 ± 0.67%; p  = 0.003; Fig.  1c ; Supplementary Data  1 ) and total, abdominal, and visceral fat mass and increased fat-free mass percentage (~2×; p  ≤ 0.030), as previously reported 15 . In addition, within-group analyses revealed a significant decrease in the reported frequency of total and lower-moderate GI symptoms (GI symptom rating score [GSRS] ≥4) over time for both IF-P and CR participants. However, when comparing the two dietary interventions at each time point, a more substantial reduction was observed in IF-P participants compared to CR participants (i.e., −9.3% vs. −5.4% and −13.2% vs. −3.9%, respectively; Table  1 ). The increased protein and lower sugar intake in IF-P compared to CR may have favorably mediated the GM and symptomatology.

a Study design with baseline participant characteristics. A registered dietitian counseled individuals from both groups each week. Time points with data collection are shown for both IF-P and CR participants. Icons created using BioRender.com. b Total daily caloric intake at each time point was not significantly different between IF-P and CR diet groups (two-sided Student’s t -test, p  < 0.05). Adjusted values are displayed by dividing total weekly intake by seven, to account for the fasting periods of IF-P. c IF-P participants lost significantly more weight over time versus CR participants. Points connected by line represent percent of weight compared to baseline weight for each participant. d Overall gut microbial colonization, as demonstrated by qPCR-based quantification of 16S rRNA gene copies per gram wet weight was unaffected by time or intervention (linear-mixed effects [LME] model, two-sided p  > 0.05). Alpha diversity metrics, e observed amplicon sequence variants (ASVs), and f Phylogenetic diversity at the ASV level significantly increased over time, independent of the intervention. g Intra-individual changes in GM community structure from baseline to weeks four and eight in IF-P participants shifted significantly throughout the IF-P intervention compared to CR as measured by the Bray-Curtis dissimilarity index (two-sided Wilcoxon rank-sum test). All box and whiskers plots display the box ranging from the first to the third quartile, and the center the median value, while the whiskers extend from each quartile to the minimum or maximum values. Heatmap of significant changes in h family- and i genus-level bacter i a by intervention. Colors indicate the within-group change beta coefficients over time for each cell, and asterisks denote significance. Black-white annotations on the bottom denote the significance of between-group change difference (by MaAsLin2 group × time interactions; p -values were corrected to produce adjusted values [ p .adj] using the Benjamini–Hochberg method). For all panels, IF-P: n  = 20, CR: n  = 19. Source data are provided as a Source Data file.

The substantial reduction in calorie intake of both groups (~40% from baseline) led us to investigate its potential impact on transient microbial colonization in the gut, as estimated by 16S rRNA gene copies (linear-mixed effects model [LME] time effect, p  = 0.114; Fig.  1d ; Supplementary Data  2 ). While it might be expected that a significant reduction in calorie intake could influence gut microbial colonization, our findings indicate that this reduction did not reach statistical significance within the timeframe of our study. This result contrasts with previous research that imposed more substantial energy restriction, such as a four-week regimen of ~800 kcal/day in participants with overweight/obesity, where overall gut microbial colonization notably decreased 4 . In addition to assessing microbial colonization, we also investigated whether the calorie reduction significantly influenced principal stool characteristics, including wet stool weight, Bristol stool scale (BSS), and fecal pH ( p  ≥ 0.066; Table  1 ). However, we did not observe statistically significant changes in these parameters over the course of the study. Moreover, there were no significant differences between the two dietary intervention groups over time (interaction effect, p  ≥ 0.051). In contrast, there were significant time effects for observed amplicon sequence variants (ASVs) and phylogenetic diversity (LME time effect, p  ≤ 0.023; Fig.  1e, f ; Supplementary Data  2 ), with values increasing at weeks four and eight compared to baseline (pairwise comparisons, p  ≤ 0.048); however, no interaction was observed for either alpha diversity metric (group × time effect, p  ≥ 0.925). To rule out the potential confounding effects of GI transit time 17 , BSS (as a surrogate marker) and stool pH were not significantly correlated with alpha diversity (Spearman correlations, p  ≥ 0.210). In relation to community composition, much of the intervention variance could be attributed to individual response upon testing nested permutational analysis of variance (PERMANOVA; R 2  = 0.749, p  = 0.001; Supplementary Table  S2 ), showcasing the highly individualistic landscape of the human GM in response to dietary intervention. However, a significant 1.8% of the variance was accounted for by the group × time interaction ( p  = 0.001). Moreover, individual responses over time showed variance between the two dietary interventions (PERMANOVA, R 2  = 0.123, p  = 0.003). This variability was apparent by assessing intra-individual differences, where a pronounced increase in Bray-Curtis dissimilarity was observed in the IF-P compared to the CR group after four (median Bray-Curtis dissimilarity, 0.53 [IQR: 0.47–0.61] vs. 0.38 [IQR: 0.33–0.47]) and eight weeks (0.50 [IQR: 0.41–0.55] vs. 0.39 [IQR: 0.33–0.45]; Fig.  1g ; Wilcoxon rank-sum test, p  ≤ 0.005).

To understand the taxa driving this GM variation from baseline to weeks four and eight between the two dietary interventions, we constructed MaAsLin2 linear-mixed models with the individual participant as a random factor 18 . We observed differential abundance patterns at the family and genus level in response to the IF-P but not the CR intervention. Of the 28 family and 69 genus-level features captured after filtering, a respective total of six and 18 taxa displayed significant interaction effects, with all significant time effects occurring from IF-P ( p .adj ≤ 0.10; Fig. 1h, i ; Supplementary Data  3 , 4 ). Notably, the changes observed at the four-week mark were more pronounced compared to those at eight weeks. These early alterations may signify an initial adaptation phase during which microbial populations respond to the modified substrate availability and nutrient composition, suggesting a degree of community resilience 19 . Increases were sustained to the third fecal collection for the family Christensenellaceae and the genera Incertae Sedis ( Ruminococcaceae family), Christensenellaceae R-7 group , and UBA1819 ( Ruminococcaceae family) (effect size > 2.0). Christensenellaceae is well regarded as a marker of a lean (anti-obesity) phenotype 20 and is associated with higher protein intake 21 . Other notable increases included Rikenellaceae , which, like Christensenellaceae , has been linked to reduced visceral adipose tissue and healthy metabolic profiles 22 , and Marvinbryantia , a candidate marker for predicting long-term weight loss success in individuals with obesity 23 . In addition, IF-P increased Ruminococcaceae , which has been noted to have an increased proteolytic and lipolytic capacity 24 . This shift in IF-P participants likely represents a change in GM substrate fermentation preferences as the diet regimen (relative protein and carbohydrate) and energy restriction is expected to increase the proteolytic: saccharolytic potential ratio 25 . In contrast, all taxa that decreased in IF-P participants were butyrate producers. These included the family Butyricicoccaceae and several genera such as Butyricicoccus (week four), Eubacterium ventriosum group (weeks four and eight), and Agathobacter (week four) (effect size < −2.0). When comparing monozygotic twin pairs, Eubacterium ventriosum group and another reduced genus, Roseburia , were more abundant in the higher body mass index (BMI) siblings 26 . Others, such as the mucosa-associated Butyricicoccus and Erysipelotricaceae UCG-003, have been positively correlated with insulin resistance and speculated to contribute to impaired glycolipid metabolism 27 .

Despite these changes in GM composition and increased fiber intake (+30% vs. baseline) of the IF-P participants 15 , we did not detect a significant shift in the abundance of the principal fecal short-chain fatty acids (SCFAs), acetate, propionate, butyrate, or valerate, as assessed by gas chromatography-mass spectrometry (GC–MS) (LME, p  ≥ 0.470; Supplementary Fig.  S1c ; Supplementary Data  5 ). Several factors likely contribute to this finding. For example, the distinct physical-chemical properties of fiber sources between IF-P and CR are inherently different. Participants adhering to the IF-P diet consumed most of their dietary fiber as liquid meal replacements (shakes) that are rich in non-digestible, oligosaccharide dietary-resistant starch 5 (RS5). In contrast, subjects on the CR regimen consumed their fiber from whole food sources such as vegetables, whole grains, and legumes. These fiber sources provided a mixture of soluble and insoluble fibers and a more complex fiber profile than IF-P participants. Moreover, even similar fiber profiles may function differently due to differences in food matrices and/or food preparation (cooking, raw consumption, etc.). Also of relevance is the timing of their fiber consumption. IF-P participants’ fiber intake was concentrated in fiber-rich shakes, offering immediate availability of fiber to the GI tract. In contrast, CR participants consumed fiber through whole foods, leading to a slower digestion and absorption process influenced by individual digestive transit times and enzymatic profiles. Interestingly, our results parallel recent work where participants more than doubled their fiber intake without affecting fecal SCFAs 28 . The disparate findings may be due to the type of dietary-resistant starch (RS) as a component of the nutrition regimen. In the current study, RS5 was included in the meal replacement shakes (eight grams/shake, two shakes/day, 16 g/day total). Prior research supports resistant starch intakes of >20 g/day favorably modulate SCFA production, primarily butyrate, over four to 12-week interventions 29 , 30 . Moreover, this lack of response in fecal SCFAs in both groups may have been further compounded by the significant reduction in energy intake in both groups, where the epithelia of the GI tract may have absorbed any potential increase in SCFAs from the dietary shift. It is worth noting that stool analysis may not be the most reliable biological surrogate for capturing SCFA flux over time 28 . Nevertheless, the changes in nutrient quality, timing, ratios, and the observed shift toward proteolytic activity suggest that the luminal matrix of digesta in the IF-P group impacted substrate availability for GM. This effect appears to be an influencing force in driving the observed beneficial shifts in microbial communities, such as Christensenellaceae and Incertae Sedis , as well as improvements in GI symptomatology in IF-P compared to CR. These results underscore the complexity of dietary influences on GM and highlight the need for further research to explore the impact of liquid meal replacements versus whole food sources on GM changes and SCFA status.

IF-P modulates circulating cytokines and gut microbiome taxa compared to CR

Caloric restriction and WL have been well known to positively influence inflammatory cytokine expression, with GM now emerging as an important modulator 31 . Surveying a panel of 14 plasma cytokines, we noted significant interaction (group × time) effects for IL-4, IL-6, IL-8, and IL-13 (LME, p  ≤ 0.034; Fig.  2a–d ; Supplementary Table  S3 ; Supplementary Data  6 ). These cytokines exhibited increases at weeks four and/or eight compared to baseline exclusively in the IF-P group (pairwise comparisons, p .adj ≤ 0.098), while no significant changes were observed in the CR group ( p .adj ≥ 0.562). Notably, IL-4 has been reported to display lipolytic effects 32 , and IL-8 has been positively associated with weight loss and maintenance 33 . Regarded as a proinflammatory myokine, IL-6 can acutely increase lipid mobilization in adipose tissue under fasting or exercise conditions 34 , 35 , 36 . IL-13 may be important for gut mucosal immune responses and is a stimulator of mucus production from goblet cells 37 , which has been recently reported to be influenced during a two-day-a-week fasting regimen in mice 38 . These results were of note considering the significant total body weight, fat, and visceral fat loss in the IF-P compared to the CR group. Surprisingly, correlational analysis with change (post – pre) in anthropometric and select plasma biomarker values with the cytokine profile did not reveal any significant associations after correcting for multiple testing effects ( p .adj ≥ 0.476; Supplementary Data  7 ). Plasma cytokines were, however, correlated with microbial composition for samples collected in the IF-P group during the intervention period (weeks four and eight) using graph-guided fused least absolute shrinkage and selection operator (GFLASSO) regression, revealing associations between cytokine-taxa pairs (Supplementary Fig.  S2a ). Of the four cytokines that increased in IF-P participants, we identified multiple significant correlations: Colidextribacter (rho = −0.55, p .adj = 0.015), Ruminococcus gauvreauii group (rho = 0.50, p .adj = 0.036), and Intestinibacter (rho = 0.45, p .adj = 0.086) with IL-4 (Supplementary Fig.  S2b ) and an unclassified genus from Oscillospiraceae (rho = −0.53, p .adj = 0.019), Colidextribacter (rho = −0.52, p .adj = 0.019), and Ruminoccus gauvreauii group (rho = 0.51, p .adj = 0.019) with IL-13 (Supplementary Fig.  S2c ).

a IL-4, b IL-6, c IL-8, and d IL-13: Each panel shows the cytokine concentration levels. Significant time effects and interaction effects (group × time) were detected using linear-mixed effects models (LME, two-sided p  < 0.05), indicating differential changes over the intervention period. IF-P participants exhibited significant increases in cytokine levels compared to baseline, as evidenced by pairwise comparisons adjusted for multiple testing using the Benjamini–Hochberg method (two-sided p .adj < 0.10). All box and whiskers plots display the box ranging from the first to the third quartile, and the center the median value, while the whiskers extend from each quartile to the minimum or maximum values. For all panels, IF-P: n  = 20, CR: n  = 19. Source data are provided as a Source Data file.

Displaying negative correlations for IL-4 and IL-13, Colidextribacter has been shown to be positively correlated to fat accumulation, insulin, and triglyceride levels in mice fed a high-fat diet 39 and positively correlated with products of lipid peroxidation, suggesting its potential role in promoting oxidative stress 40 . Conversely, Ruminoccus gauvreauii group was positively correlated with IL-4 and IL-13. Although limited information is available regarding the host interactions of this microbe, this genus is considered a commensal part of the core human GM and able to convert complex polysaccharides into a variety of nutrients for their hosts 41 . While these findings highlight the potential interplay between specific microbes and cytokine profiles, the directional influence—whether microbial changes drive cytokine alterations or vice versa—cannot be determined in this study setting. Furthermore, despite the change in cytokine profiles in the IF-P group, we did not detect any significant time or group × time effects when measuring lipopolysaccharide-binding protein (LBP; Δ pre/post, IF-P: 0.24 ± 0.31 vs CR: −0.93 ± 0.49 μg/mL; p  ≥ 0.254), a surrogate marker for gut permeability 42 . While the GM plays a crucial role in modulating the gut-immune axis, the observed cytokine fluctuations and microbial associations might also involve other factors. These include the production of specific metabolites due to shifts in microbial composition as well as the influence of the dietary regimen itself, which may have a central role in shaping these interactions.

IF-P and CR yield distinct circulating metabolite signatures and convergence of multiple metabolic pathways

To understand the potential differential impact of IF-P versus CR on the host, we surveyed the plasma metabolome, reliably detecting 136 plasma metabolites across 117 samples (i.e., QC CV < 20% and relative abundance > 1000 in 80% of samples). Based on outlier examination (random forest [RF] and principal component analysis [PCA]), no samples were categorized as outliers, and all data were retained for subsequent analysis. Metabolomic profile shifts were observed in both IF-P and CR groups compared with baseline (Canberra distance), however, these did not differ significantly by group or time (weeks four and eight; Wilcoxon rank-sum test, p  ≥ 0.087; Supplementary Fig.  S3a ). We prepared a general linear model (GLM) with age, sex, and time as covariates and corrected for false discovery rate (FDR). When controlling for these relevant covariates, we observed significant differences between IF-P and CR for 15 metabolites (Fig.  3a , Supplementary Table  S4 ): 2,3-dihydroxybenzoic acid, malonic acid, choline, agmatine, protocatechuic acid, myoinositol, oxaloacetic acid, xylitol, dulcitol, asparagine, n-acetylglutamine, sorbitol, cytidine, acetylcarnitine, and urate ( p .adj ≤ 0.089). To estimate the univariate classification performance of the 15 significant metabolites, we performed a receiver operating characteristic (ROC) analysis. Ten metabolites demonstrated a moderate area under the curve (AUC) (0.718–0.819), while five metabolites had an AUC < 0.70. Therefore, to improve classification performance, we constructed a supervised PLS-DA model using levels of the 15 significant metabolites ( p .adj ≤ 0.089) and analyzed variable importance in projection (VIP) scores (Supplementary Fig.  S3b ). Five metabolites with a VIP > 1.0 (2,3-dihydroxybenzoic acid, malonic acid, protocatechuic acid, agmatine, and myoinositol) were retained to construct an enhanced orthogonal projection to latent structures discriminant analysis (OPLS-DA) model. In contrast, the model fit was assessed with 100-fold leave-one-out cross-validation (LOOCV; see “Methods” section). Permutation testing showed the refined OPLS-DA model to have an acceptable fit to data ( Q 2  = 0.460, p  < 0.001), with appreciable explanatory capacity ( R 2  = 0.506, p  < 0.001; Supplementary Fig.  S3c ). The ROC analysis produced an area under the curve (AUC) of 0.929 (95% CI: 0.868–0.973, sensitivity = 0.8, specificity = 0.9; Supplementary Fig.  S3d ) between the CR and IF-P groups showing good accuracy of the GLM and providing strong support for the differential expression of these 15 metabolites between groups.

a Abundance and log fold-change of significant plasma metabolites between IF-P and CR groups as determined by a general linear model (GLM) adjusted for age, sex, and time. All GLM analyses utilized two-sided p -values, with multiple testing corrections applied using the Benjamini–Hochberg method ( p .adj). Metabolome pathway analysis was conducted for b IF-P and c CR using all reliably detected metabolites showing significantly altered pathways ( p .adj < 0.10) with moderate and above impact (>0.10). Impact scores were calculated using a hypergeometric test, while significance was assessed via a test of relative betweenness centrality, emphasizing the changes in metabolic network connectivity. For all panels, IF-P: n  = 20, CR: n  = 19. Source data are provided as a Source Data file.

Two metabolites, malonic acid, and acetylcarnitine, increased compared to the CR intervention. Several other investigators have noted the increase in acetylcarnitine via fasting protocols 43 , 44 . This increase is consistent with free fatty acid mobilization and increased transportation of these fatty acids via carnitine acylation into the mitochondria for fatty acid oxidation. These results would also be consistent with the expected ketogenesis, although not documented in our study, but noted by similar fasting interventions 44 . Relatedly, malonic acid, a naturally occurring organic acid, is a key regulatory molecule in fatty acid synthesis via its conversion to acetoacetate; hence, our results may reflect this increased synthesis in response to the mobilization and oxidation of fatty acids occurring during fasting. Other metabolites that decreased with IF-P include several sugar alcohols (myoinositol, dulcitol, and xylitol). Dulcitol (galactitol) is a sugar alcohol derived from galactose. It is possible that during fasting, levels of dulcitol decrease as glucose (initially) and free fatty acids (after 24–36 h of fasting) are preferentially utilized as energy substrates. One amino acid (asparagine) and one amino acid analog (N-acetylglutamine, associated with consumption of a Mediterranean diet 45 ) also decreased with IF-P relative to CR. Finally, 2,3-dihydroxybenzoic acid significantly decreased with IF-P. This metabolite is formed during the metabolism of flavonoids, as it is found abundantly in fruits, vegetables, and some spices. At the cellular level, this hydroxybenzoic acid functions as a cell signaling agent and has been speculated as a potentially protective molecule in various cancers 46 . It is unclear whether this metabolite decreased due to either dietary intake or metabolic processes related to high-protein intake or the fasting protocol. Collectively, the metabolic responses to these dietary regimens reflect the interrelationships of several anabolic and catabolic physiologic responses to three key components of these interventions: (a) the WL process itself, (b) changes in amount (and type) of macronutrient distribution (i.e., meal replacement shakes vs. whole food diet approach; higher vs. normal protein intakes), and (c) the adherence to fasting (IF-P only).

To determine the significantly impacted pathways of the dietary interventions, we grouped participant samples according to baseline or intervention period (weeks four and eight), with IF-P and CR assessed separately. A total of 14 pathways were significant in the IF-P group ( p .adj < 0.10; Fig.  3b ), with three displaying large impact coefficients (>0.5): (1) Glycine, serine, and threonine metabolism, (2) alanine, aspartate, and glutamate metabolism, and (3) ascorbate and aldarate metabolism. In comparison, 24 pathways were significant for the CR group (Fig.  3c ), with four showing large impact coefficients (>0.5): (1) Phenylalanine, tyrosine, and tryptophan biosynthesis, (2) alanine, aspartate, and glutamate metabolism, (3) citrate cycle (TCA cycle), and (4) glycine, serine and threonine metabolism. Notably, the glycine, serine, and threonine pathway has recently been found in preclinical models to play a pivotal role in longevity and related life-sustaining mechanisms independent of diet, though heavily impacted by fasting time and caloric restriction 47 . This may be partially related to the ability of glycine to increase tissue glutathione 48 , 49 and protect against oxidative stress 50 . In our analysis, this pathway was significant in both diet groups and is biochemically and topologically related to the additionally captured amino acid pathway, alanine, aspartate, and glutamate metabolism, as well as the energy-releasing pathway, the citrate cycle (TCA cycle). Notably, in the CR group, phenylalanine, tyrosine, and tryptophan biosynthesis, are important for neurotransmitter production and reported to be suppressed (tryptophan) in obesity 51 . This representation may have also been attributed to the differences in protein intake 52 or differences in dietary diversity 53 , yet to be determined. Regardless, we noted similar representations of pathway impact between IF-P and CR, with metabolic response centered on utilization of amino acids in addition to lipid turnover and energy pathways.

Gut microbiome and plasma metabolome latent factors indicate differential multi-omic signatures between IF-P and CR regimens

As the plasma metabolome has been suggested as a bidirectional mediator of GM influence on the host 54 , we performed a multi-omics factor analysis (MOFA) 55 to identify potential patterns of covariation and co-occurrence between the microbiome and circulating metabolites. Operating in a probabilistic Bayesian framework, MOFA simultaneously performs unsupervised matrix factorization to obtain overall sources of variability via a limited number of inferred factors and identifies shared versus exclusive variation across multiple omic data sets 55 . Eight latent factors were identified (minimum explained variance ≥2%; see “Methods” section), with the plasma metabolome and GM explaining 37.12% and 17.49% of the overall sample variability, respectively (Fig.  4a ). Based on significance and the proportion of total variance explained by individual factors for each omic assay, Factors 1 ( R 2  = 11.98) and 6 ( R 2  = 5.28) captured the greatest covariation between the two omic layers (Fig.  4a ; Supplementary Table  S5 ). In contrast, Factors 2 and 5 were nearly exclusive to the metabolome, and factors 3 and 4 to the GM. Interestingly, Factor 1 was significantly negatively correlated to dietary protein intake (Spearman rho = −0.270, p.adj = 0.021; Fig.  4b ) and captured the variation associated with the CR diet (Wilcoxon rank-sum test, p .adj = 3.2e-04; Fig.  4c ). Factor 6 had the greatest number of significant correlations, including negative associations with visceral adipose tissue, waist circumference, body weight, BMI, fat mass, android fat, subcutaneous adipose tissue, dietary sodium, carbohydrate, fat, energy intake (kcal), and sugar (Spearman rho ≤ −0.220, p .adj ≤ 0.075) and captured the variation associated with IF-P (Wilcoxon rank-sum test, p .adj = 0.007).

a The cumulative proportion of total variance explained ( R 2 ) and proportion of total variance explained by eight individual latent factors for each omic layer. b Spearman correlation matrix of the eight latent factors and clinical anthropometric and dietary covariates. Each circle represents a separate association, with the size indicating the significance (-log10 ( p -values)) and the color representing the effect size (hue) with its direction (red: positive; blue: negative). All correlations are calculated using two-sided tests. Asterisks within a circle denote significance after adjustment with the Benjamini–Hochberg method. c Scatter plot of Factors 1 and 6, with each dot representing a sample colored by intervention. Box and whisker plots illustrate significant differences between groups after adjusting for multiple testing using the Benjamini–Hochberg method (Wilcoxon rank-sum test; top = Factor 1, p .adj = 3.2e-04; right = Factor 6, p .adj = 0.007). The plots show boxes ranging from the first to the third quartile and the median at the center, with whiskers extending to the minimum and maximum values. d Factor 1 and 6 loadings of genera and metabolites with the largest weights annotated. Symbols: * p .adj < 0.10, ** p .adj < 0.01, *** p .adj < 0.001, **** p .adj < 1.0e-04. For all panels, IF-P: n  = 20, CR: n  = 19. Source data are provided as a Source Data file.

Assessing the positive weights (feature importance) of Factor 1 revealed a microbial and metabolomic signature linked with CR, including the taxa Faecalibacterium , Romboutsia , and Roseburia , and the plasma metabolites myoinositol, agmatine, N-acetylglutamine, erythrose, and mucic acid (Fig.  4d ). Previous dietary restriction studies have reported co-occurrence of gut microbial taxa and plasma metabolites that span a wide variety of applications and investigations 56 . The specific co-occurrences observed in Factor 1 exhibited an abundance of butyrate-producing bacterial taxa that utilize carbohydrates as their predominant substrate and plasma metabolites that are generally involved in carbohydrate metabolism, such as erythrose, an intermediate in the pentose phosphate pathway (PPP), and mucic acid which is derived from galactose and/or galactose-containing compounds (i.e., lactose). These co-occurrence patterns biologically cohere considering the nutritional profile of the CR group and the large contribution of fiber-rich, unrefined carbohydrates and reduction in sugar (~50% kcal from sugar). Indeed, these nutritional changes may have influenced the GM to accommodate changes in dietary substrate more efficiently. One interesting co-occurrence was the genus Romboutsia and metabolite N-acetylglutamine. Romboutsia has been shown to produce several SCFAs and ferment certain amino acids, including glutamate 57 . N-acetylglutamine is biosynthesized from glutamate; thus, its co-occurrence with the abundance of Romboutsia encourages further exploration into this interaction 58 .

Factor 6 captured the signature associated with IF-P, with positive contributions from the taxa Incertae Sedis ( Ruminococcaceae family), Erysipelatoclostridium , Christensenellaceae R-7 group , Oscillospiraceae UCG-002, and Alistipes , and the plasma metabolites malonic acid, adipic acid, succinate, methylmalonic acid, and mucic acid (Fig.  4d ). Prior work has established that Alistipes increases from diets rich in protein and fat, and contributes to the highest number of putrefaction pathways (i.e., fermentation of undigested proteins in the GI tract) over the other commensals 59 . This could explain the co-occurrence of plasma metabolites from protein catabolism, such as 2-aminoadipid acid, adipic acid, and glutamic acid 22 , 59 . Oscillospiraceae has recently been viewed with next-generation probiotic potential, harboring positive regulatory effects in areas related to obesity and chronic inflammation 60 . Mentioned prior, recent studies have reported on the role of Christensenellaceae on human health, participating in host amino acid and lipid metabolism as well as fiber fermentation 20 , with Christensenellaceae R-7 group notably evidenced to correlate with visceral adipose tissue reduction 22 . As such, the elevated abundance of microbes in the GM of IF-P participants observed in this study in tandem with the co-occurrence of metabolites indicative of protein degradation and mobilization and oxidation of fatty acids, such as methylmalonic acid, malonic acid, and succinate, presents a nascent multi-omic signature of IF-P. In addition, and more pronounced in the IF-P vs CR group, participants decreased sugar intake by ~75% (kcals) compared to baseline levels. Considering the other regimental components of IF-P, the differences in multi-omic signatures likely display the selective pressures of these two interventions.

Gut microbiome (GM) composition is associated with weight loss (WL) responsiveness to IF-P diet

The IF-P intervention produced a microbiome and metabolomic response; however, the loss in body weight and fat across individuals varied (Fig.  5a ). To provide deeper characterization and explore differential features of WL responsiveness, we performed a GM-focused subgroup analysis by employing shotgun metagenomic and untargeted fecal metabolomic surveys in 10 individuals that either achieved ≥10% loss in body weight or bordered on clinically important WL (i.e., >5% BW; herein, ‘High’ and ‘Low’ responders) 61 . Importantly, baseline characteristics between WL responder classification did not differ significantly (baseline body weight: High, 108.9 ± 30.8 vs. Low, 81.9 ± 18.1 kg, p  = 0.117; Supplementary Table  S6 ). Assessing the GM at the fundamental taxonomic rank, species composition showed significant separation by weight loss response evaluated by Bray-Curtis dissimilarity (group × time: R 2  = 0.114, p  = 0.001; Fig.  5b ; Supplementary Table  S7 ), with most of the variation explained by the individual ( R 2  = 0.711, p  = 0.001). In comparison, species level alpha diversity did not differ significantly between classifications (group × time: p  ≥ 0.674; Fig.  5c, d ). Identifying 212 species after filtering, we noted significant differences in bacterial abundances between groups over time (Fig.  5e ; Supplementary Data  8 ). A total of 10 features increased in the High-responder group relative to the Low-response group over the eight-week study period, including Collinsella SGB14861 , Clostridium leptum , Blautia hydrogenotrophica , and less typified species; GGB74510 SGB47635 (unclassified Firmicutes), GGB3511 SGB4688 (unclassified Firmicutes), Faecalicatena contorta , Lachnospiraceae bacterium NSJ-29 , Phascolarctobacterium SGB4573 , GGB38744 SGB14842 (unclassified Oscillospiraceae ), and Massiliimalia timonensis (effect size ≥ 1.163, p .adj ≤ 0.092). The increase in Collinsella , a less characterized anaerobic pathobiont that produces lactate and has been associated with low-fiber intakes 62 , 63 and lipid metabolism 64 , may have been related to the periods of CR and IF, in conjunction with the greater influx of host-released fatty acids in the High-responder group. Relatedly, Clostridium leptum growth has been linked with increases in monounsaturated fat intake, reductions in blood cholesterol 65 , and stimulation of Treg induction (i.e., anti-inflammatory) 66 . The latter association is relevant to the SCFA-promoting (primarily butyrate) qualities of Clostridium leptum 67 . Blautia hydrogenotrophica , an acetogen with bidirectional metabolic cross-feeding properties (e.g., transfer of hydrogen and acetate), is also important for butyrate formation 68 . Taxa that decreased relative to the Low-responder group; Eubacterium ventriosum , Streptococcus salivarius , Eubacterium rectale , Anaerostipes hadrus , Roseburia inulinivorans , Mediterraneibacter glycyrrhizinilyticus , and Blautia massiliensis (effect size ≤ −1.690, p .adj ≤ 0.078), included butyrate producers, Eubacterium ventriosum , Eubacterium rectale , Roseburia inulinivorans , and others, such as Streptococcus salivarius , a nuclear factor kappa B (NF-κB) activity repressor 69 and Peroxisome proliferator-activated receptor gamma (PPARγ) inhibitor potentially influencing lipid and glucose metabolism 70 . Investigating monozygotic (MZ) twin pairs, Eubacterium ventriosum was more abundant in the higher BMI siblings 26 , with enhanced scavenging fermentation capabilities 71 . Roseburia inulinivorans is a mobile firmicute (flagella) that harbors a wide-ranging enzymatic repertoire able to act on various dietary polysaccharide substrates suggestive of the ability to respond to the availability of alternative dietary substrates 72 . While we noted a more variable shift in fecal total SCFAs, acetate, propionate, butyrate, or valerate (via targeted GC–MS), in the Low weight loss responders, there was no significant difference when compared to High weight loss responders (Wilcoxon rank-sum test, p  ≥ 0.210; Supplementaryl Fig.  S4a ; Supplementary Data  9 ).

a Relative weight loss over the eight-week intervention for each participant in the IF-P group. b NMDS ordination showed the personalized trajectories of participants’ microbiomes over time. Dotted lines connect the same individual and point toward the final sample collection. No significant time or group × time interaction effects for alpha diversity metrics, c observed species, and d the Shannon index. Box and whiskers plots display the box ranging from the first to the third quartile, and the center the median value, while the whiskers extend from each quartile to the minimum or maximum values. Volcano plots displaying differential abundance between High and Low weight loss responders for e microbial species and f functional pathways. Significant features were more enriched in High and Low weight loss responders colored orange and light blue, respectively. g Alluvial plot displaying the fecal metabolite profile at the subclass level (Human Microbiome Database). Most abundant metabolite subclasses displayed (i.e., ≥1%). Metabolome pathway analysis for h High and i Low weight loss responders using all reliably detected fecal metabolites showing altered pathways with moderate and above impact (>0.10). Impact was calculated using a hypergeometric test, while significance was determined using a test of relative betweenness centrality. j Grid-fused least absolute shrinkage and selection operator (GFLASSO) regression of species from differential abundance analysis displayed correlative relationships with fecal metabolites. Species with greater abundance in High (High > Low) and Low (Low > High) weight loss responders are separate‘. For all panels, High: n  = 5, Low: n  = 5. Source data are provided as a Source Data file.

Less affected compared to taxonomic features were the 275 microbial-affiliated metabolic pathways identified after filtering, of which gluconeogenesis III and guanosine ribonucleotides de novo biosynthesis were increased (effect size ≥ 0.108, p .adj = 0.079), while super pathway of L-alanine biosynthesis, sucrose degradation IV (sucrose phosphorylase), sucrose degradation III (sucrose invertase), super pathway of thiamine diphosphate biosynthesis III, and flavin biosynthesis I (bacteria and plants) were decreased in the High relative to the Low weight loss responder group (effect size ≤ −0.247, p .adj ≤ 0.079; Fig.  5f ; Supplementary Data  10 )

As the difference in microbial shifts versus function is well established, we also tracked the fecal metabolome to better understand metabolic modification/production and identify potential microbial metabolic targets for future weight loss interventions. Overall, we reliably detected (QC relative standard deviation > 20% and mean intensity value > 1000 in 80% of samples) and annotated 607 (Human Metabolome Database) compounds across fecal samples. Notably, we found the fecal metabolite profile of both subgroups abundant in amino acids, peptides, and analogs, with decreases in sulfates, furanones, and quaternary ammonium salts and increases in cholestane steroids, carboxylic acid derivatives, and imidazoles (Fig.  5g ). Assessing metabolite changes between groups did not yield significance when comparing logFC values (Wilcoxon rank-sum test, p .adj > 0.10; Supplementary Fig.  S4b ). Pathway analysis of High weight loss responders revealed prominent metabolic signatures relevant to lipid metabolism (glycerolipid and arachidonic metabolism), nucleotide turnover (pyrimidine metabolism), and aromatic amino acid formation (phenylalanine, tyrosine, and tryptophan biosynthesis; Fig.  5h , Supplementary Data  11 ). In comparison, the more prominent enriched pathways for Low weight loss responders included those related to amino acid and peptide metabolism (glycine, serine, and threonine, d-glutamine and d-glutamate, and tyrosine metabolism and arginine biosynthesis; Fig.  5i , Supplementary Data  12 ).

Finally, species captured by our differential abundance analysis were channeled into a GFLASSO model with the fecal metabolome library to select metabolically relevant compounds best predicted by microbial abundances. Restricting taxa and metabolites displaying stronger co-occurrence signals (GFLASSO coefficients > 0.02), we noted several patterns (Fig.  5j ). This included positive associations between GGB3511 SGB4688 (unclassified Firmicute) and malonic acid (important to fatty acid metabolism), as well as Roseburia inulinivorans and 3-Hydroxy-2-oxo-1H-indole-3-acetic acid. Negative associations included Phascolarctobacterium SGB4573 with the fatty acid ester, methyl sorbate, and Streptococcus salivarius (anti-inflammatory) with leukotriene B4 dimethylamide.

Differences detected in our subgroup analysis suggest that the GM composition plays a role in WL responsiveness during IF-P interventions. Notable differences in taxa and fecal metabolites suggest differing substrate utilization capabilities and nutrient-acquiring pathways between High and Low responders, despite being on the same dietary regimen. Although differences between High and Low responders were statistically significant for the microbiome data, the magnitude of differences varied, suggesting further research is needed to clarify these differences.

Long-term IF-P remodels the gut microbiome after substantial weight loss – A case study

Considering the microbiomic and metabolic importance of sustained WL, we additionally performed a longitudinal, exploratory case study analysis on the participant who lost the most body weight during the eight-week WL period (−15.3% BW, −24.9 kg). Under rigorous clinical supervision, this individual was guided through and comprehensively tracked over 52 weeks, strictly adhering to an IF-P regimen, including WL (0–16 weeks) and maintenance (16–52 weeks) periods, which included adjusting the calorie intake to maintain energy balance. Microbial richness and evenness at the species level displayed a general inverse trend with body weight reduction, although they converged at 52 weeks (Fig.  6a, b ). Species dissimilarity peaked at weeks four and 16, after which it plateaued, but remained consistently higher in comparison to baseline over the 52-week period (Fig.  6c ). Examining positive linear coefficients of a PERMANOVA model, constructed to detect variation between community compositions over time, dominant influences included several species within the Lachnospiraceae family such as Fusicatenibacter saccharivorans , Blautia wexlerae , Blautia massillensis , Anaerostipes hadrus , and Coprococcus comes and others like Akkermansia muciniphila (Fig.  6d ). Negative contributions included species from the Oscillospiraceae family, such as Ruminococcus bromii and Ruminococcus torques . Indeed, visualizing community composition over the sampling time points suggested specific GM remodeling (Fig.  6e ; Supplementary Data  13 ). Many keystone taxa prominent over time in the microbiome are highly relevant to the significant reduction in body weight and metabolic improvement of the case-study participant. For example, Blautia wexlerae , a commensal bacterium recently reported to confer anti-adipogenesis and anti-inflammatory properties to adipocytes 73 became visually more prominent over time. This association was also the case for the health-associated microbe, Anaerostipes hadrus , which converts inositol stereoisomers (including myoinositol) to propionate and acetate, apt to improve insulin sensitivity and reduce serum triglyceride levels 74 , translating to reduced host metabolic disease risk 75 . Other elevated taxa, like the mucin-degrading Akkermansia muciniphila and Bacteroides faecis , are negatively correlated with markers for insulin resistance 76 . There was also a notable bloom of Collinsella SGB14861 (anaerobic pathobiont producing lactate) 63 and suppression of Eubacterium rectale , Ruminococcus torques (associated with circadian rhythm disruption in mice) 77 , and Ruminococcus bromii (an exceptional starch degrader) 78 .

Change in alpha diversity metrics a observed species and b Shannon index with percentage of baseline body weight. c Bray-Curtis dissimilarity at the species level with d top PERMANOVA model coefficients (analysis: species~time). e Alluvial plot displaying the variation in abundance of the 20 most prevalent bacteria over time. For visual clarity, the less abundant taxa are not displayed. f Canberra distance of fecal metabolome with g top PERMANOVA model coefficients (analysis: pathway~time). h Pathway analysis of fecal metabolites comparing baseline to subsequent sample collections. Data are plotted as -log10(p) versus pathway impact. Node size corresponds to the proportion of metabolites captured in each pathway set, while node color signifies significance. Impact was calculated using a hypergeometric test, while significance was determined using a test of relative betweenness centrality. No p -value adjustments were made. Source data are provided as a Source Data file.

Compared to the more pronounced shifts in the GM, an inspection of Bray-Curtis dissimilarity at the microbial metabolic pathway level was much less affected (Supplementary Fig.  S5a ). Though positive contributions in multiple biosynthesis pathways were noted, as well as reductions in the superpathway of UDP-glucose-derived O-antigen building blocks biosynthesis and glucose and glucose-1-phosphate degradation (Supplementary Fig.  S5b ; Supplementary Data  14 ). We also tracked the fecal metabolome concordance with the GM to corroborate potential metabolic output. Shifts in metabolites captured by calculating the Canberra distance were prominent (Fig.  6f ), with positive influences from agrocybin (possessing antifungal activity 79 ), nicotinic acid (nicotinamide adenine dinucleotide precursor), and sulfate, and reductions in cadaverine (involved in the inhibition of intestinal motility 80 ), maltitol, acetohydroxamic acid (a urease inhibitor), and hypoxanthine, after removing the dominant amino acid subclass (Fig.  6g ; Supplementary Fig.  S5c ). At the chemical class level, we observed apparent shifts in chemical subclasses; cholestane steroids, amines, purines, and purine derivatives, and amino acids, peptides, and analogs (Supplementary Fig.  S5d ). Given our case-study approach, we performed a pathway analysis using all reliably detected fecal metabolites at each collection point over 52 weeks. Pathway analysis (Fig.  6h ) identified primary bile acid biosynthesis ( p  = 0.014) and cysteine and methionine metabolism ( p  = 0.096) as having the greatest significance, while the greatest impact (I) was observed in phenylalanine, tyrosine, and tryptophan biosynthesis and linoleic acid metabolism ( I  = 1.0). Alanine, aspartate, and glutamate metabolism ( I  = 0.756), vitamin B6 metabolism ( I  = 0.647), sulfur metabolism ( I  = 0.532), phenylalanine metabolism (I =  0.357), and nicotinate and nicotinamide metabolism ( I  = 0.194) also displayed marked pathway impacts (Supplementary Fig.  S5e ; Supplementary Data  15 ). Together, these integrated findings from the group comparisons (IF-P vs. CR), high vs. low responders, and the case study, suggest that the remodeling of the gut microbiome through sustained weight loss on an IF-P regimen not only alters the microbial composition but also influences key metabolic pathways and output, reflective of fat mobilization and metabolic improvement.

Our study demonstrates distinct effects of IF-P on gut symptomatology and microbiome, as well as circulating metabolites compared to continuous CR. We observed significant changes in the GM response to both interventions; however, the IF-P group exhibited a more pronounced community shift and greater divergence from baseline (i.e., intra-individual Bray-Curtis dissimilarities). This shift was characterized by increased specific microbial families and genera, such as Christensenellaceae , Rikenellaceae , and Marvinbryantia , associated with favorable metabolic profiles. Furthermore, IF-P significantly increased circulating cytokine concentrations of IL-4, IL-6, IL-8, and IL-13. These cytokines have been linked to lipolysis, WL, inflammation, and immune response. The plasma metabolome analysis revealed distinct metabolite signatures in IF-P and CR groups, with the convergence of multiple metabolic pathways. These findings shed light on the differential effects of IF regimens, including IF-P as a promising dietary intervention for obesity management and microbiotic and metabolic health.

While acknowledging individual contributions of WL, protein pacing, and IF, we propose that the beneficial shifts observed may be best characterized as the culmination of features inherent in our IF-P approach. For example, it is possible that microbial competition is leveraged during reduced and intermittent nutritional input periods, emphasizing nutrient composition and food matrix type (combination of whole food and meal replacements vs. primarily whole food), affecting available substrates for gut microbes. IF-P participants’ fiber intake was concentrated in fiber-rich (RS5 type) shakes, offering immediate availability of fiber to the GI tract. In contrast, CR participants consumed fiber through whole foods, leading to a slower digestion and absorption process influenced by individual digestive transit times and enzymatic profiles. This nutritional environment may create ecological niches that support symbiont microbial communities. In this investigation, we provide support of such remodeling, with intentional fasting and increased relative protein (protein pacing) consumption well-validated to improve body composition and metabolism during weight loss 7 , 8 , 15 . Our results align with previous studies on CR, where greater relative protein intake was associated with an increased abundance of Christensenella 81 . This increase is likely a result of increased amino acid-derived metabolites 21 . We also observed increased signatures of amino acid metabolism in the GM of IF-P participants, which may be attributed to increased nitrogen availability, prompting de novo amino acid biosynthesis. The liquid format of two of the daily meals and precise timing of high-quality protein consumption (Protein Pacing) in the IF-P regimen may have influenced these results, as amino acids play essential roles in microbial communities, acting as energy and nitrogen sources and essential nutrients for amino acid auxotrophs.

In addition to the differences in nutrient composition, the IF-P group exhibited a profound reduction (33%) in visceral fat 15 . This reduction is significant because visceral fat is highly correlated with GM. While the specific influence of GM on fat depots in our study remains unclear, the shift in cytokine profile and metabolic pathways suggests an interaction between GM and fat metabolism. Regarding GM-host interaction, we did not detect changes in gut permeability assaying LBP. However, correlations were found with cytokines IL-4 and IL-13 and microbes Colidextribacter (negative association) and Ruminoccus gauveauii group (positive association). These associations may reflect the direct impact of the dietary intervention, yet they also hint at a deeper crosstalk within the gut-immune axis. This crosstalk is known to play a pivotal role in modulating host inflammation and influencing adipose tissue signaling pathways 42 . Furthermore, the observed microbial shifts, including changes in populations of Christensenella , suggest a nuanced role for certain microbes in regulating metabolic health. Notably, certain strains of Christensenella have been implicated in the regulation of key metabolic markers, such as glycemia and leptin levels, and in promoting hepatic fat oxidation 82 .

Our findings also underscore that GM composition plays a role in WL responsiveness during IF-P interventions. Subgroup analysis based on WL responsiveness revealed significant differences in species composition at the taxonomic level. The High-responder group showed an increased abundance of certain bacteria associated with metabolic benefits and anti-inflammatory effects. In contrast, the Low-responder group exhibited an increased abundance of butyrate-producing and nutritionally adaptive species (e.g., Eubacterium ventriosum 71 and Roseburia inulinivorans 72 ). Fecal metabolome analysis further highlighted differences between the two subgroups, with distinct metabolic signatures and enrichment in specific metabolic pathways. Notably, the High WL responders displayed enrichment of fecal metabolites involved in lipid metabolism. In contrast, Low responders were more prominent in pathways related to the metabolism of amino acids and peptides, including glycine, serine, and threonine, d-glutamine, and d-glutamate, as well as tyrosine metabolism and arginine biosynthesis. The latter metabolic signature has been reported in individuals with severe obesity undergoing high-protein, low-calorie diets 83 . As both High and Low WL responders were consuming the same diet, our results suggest differences in GM composition and metabolism, which could play a role in determining the success of an IF-P regimen. Though, as these enrichment analyses were performed in an exploratory manner, we acknowledge the need for a more systematic approach to validate these findings.

Finally, we provide evidence of long-term GM stabilization from these changes by following one individual over 12 months. Dietary restriction is widely used to reduce fat mass and weight in individuals with or without obesity; however, weight regain after such periods presents a critical challenge, and the underlying homeostatic mechanisms remain largely elusive. Notably, keystone taxa that became more prominent over time were associated with anti-adipogenesis, improved insulin sensitivity, and reduced metabolic disease risk. The microbial shifts were accompanied by noticeable changes in the fecal metabolome, with shifts in various metabolites and chemical subclasses. Pathway analysis identified impacts on primary bile acid biosynthesis, cysteine and methionine metabolism, and other fat mobilization and metabolic improvement pathways. These shifts were accompanied by noticeable changes in the fecal metabolome, particularly in metabolites and chemical subclasses related to lipid metabolism, nucleotide turnover, and aromatic amino acid formation.

Despite the valuable insights from our study on the complex interactions between intermittent fasting, higher protein intake using protein pacing, the GM, and circulating metabolites in obese individuals, several limitations should be acknowledged. First, our reliance on fecal samples to represent the GM may have overlooked potential microbial populations in the upper GI tract. Including samples from proximal regions in future studies would provide a more comprehensive understanding of the gut microbiome’s response to IF-P and CR. In addition, the sample size for our study was determined based on the primary outcomes related to body weight and composition from the parent study 15 . This sample size may have reduced statistical power and potentially amplified individual variability among participants. However, it is important to note that the smaller RCT design allowed for more precise control over diet and lifestyle factors, minimizing potential confounding influences on the study outcomes. Furthermore, the study’s duration was limited to eight weeks, which prevented potential insights into the differential long-term effects between the two interventions. However, we were able to extend the follow-up duration and conduct periodic assessments for a year in our case-study participant, offering a more comprehensive understanding of the sustainability of the observed changes and the potential for weight regain for IF-P. The current study compared a combination of whole food and supplements (shakes and bars; IF-P) versus primarily whole food (CR), which together with variations in protein and fiber content and type may have influenced the gut symptomatology and nutrient absorption between groups. Additionally, study participants self-reported dietary intake daily, although there was close monitoring of intake through the return of empty food packaging/containers of consumed food and daily monitoring by investigators and weekly meetings with a registered dietitian. Overall, knowledge gaps are present in this research, including how the microbiome is rebuilt after food reintroduction and how overall caloric restriction and specific macronutrients contribute to this process. However, considering the multifactorial nature of weight loss and metabolic health, our work represents an important precedent for future work. Future investigators should consider integrating these factors to provide a more comprehensive understanding of the underlying mechanisms. Additional research is warranted to characterize the metabolic signature of IF-P, the time relationship between these fasting periods, and the analysis of these metabolic changes. A strength of our High-Low-responder and case-study analyses is the hypothesis-driving nature of the findings, from which targeted microbiome and/or precision nutrition interventions can be designed and tested.

In conclusion, our study provides valuable insights into the complex interactions among intermittent fasting and protein pacing, the GM, and circulating metabolites in individuals with obesity. Specifically, intermittent fasting - protein pacing significantly reduces gut symptomatology and increases gut microbes associated with a lean phenotype ( Christensenella ) and circulating cytokines mediating total body weight and fat loss. These findings highlight the importance of personalized approaches in tailoring dietary interventions for optimal weight management and metabolic health outcomes. Further research is necessary to elucidate the underlying mechanisms driving these associations and to explore the therapeutic implications for developing personalized strategies in obesity management. Additionally, future studies should consider investigating microbial populations in upper GI sections and potential intestinal tissue remodeling to gain a more comprehensive understanding of the gut microbiome’s role in these interventions.

Study design and participants

The protocol of the clinical trial was registered on March 6, 2020 (Clinicaltrials.gov; NCT04327141), and the results of the primary analysis have been published previously 15 . Briefly, participants were recruited from Saratoga Springs, NY, and were provided informed written consent in accordance with the Skidmore College Human Subjects Institutional Review Board before participation (IRB#: 1911-859), including consent for the use of samples and data from the current study. Each procedure performed was in adherence with New York state regulations and the Federal Wide Assurance, which follows the National Commission for the Protection of Human Subjects of Biomedical and Behavioral Research, and in agreement with the Helsinki Declaration (revised in 1983). Their physicians performed a comprehensive medical examination/history assessment to rule out any current cardiovascular or metabolic disease. For at least six months before the start of the study, all eligible participants were either sedentary or lightly active (<30 min, two days/week of organized physical activity), with overweight or obesity (BMI > 27.5 kg/m2; % body fat > 30%), weight stable (±2 kg), and middle-aged (30–65 years). In addition, participants taking antibiotics, antifungals, or probiotics within the previous two months were excluded. Enrolled participants were matched for body weight, BMI, and body fat and randomly assigned to one of two groups: (a) IF-P ( n  = 21; 14 women; 7 men) or (b) CR ( n  = 20; 12 women; 8 men) for eight weeks. During a one-week run-in period, subjects maintained a stable body weight by consuming a similar caloric intake as their pre-enrollment caloric intake while maintaining their sedentary lifestyle. This was confirmed by matching their pre-enrollment dietary intake to the one-week run-in diet period 15 . Following baseline testing, participants were provided detailed instructions on their weight loss dietary regimen (Supplementary Table  S1 ) and received weekly dietary counseling and compliance/adherence monitoring from the research team via daily food records, and weekly registered dietitian meetings, along with weekly visits to the Human Nutrition and Metabolism laboratory at Skidmore College (Saratoga Springs, NY) for meal distribution and empty packet/container returns. All outcome variables were assessed pre (week 0), mid (week 4), and post (week 8). All participants were compensated $100 for successful completion of the study and received an additional monthly stipend of $75 for groceries (CR group only) or up to two meals per day of food supplements and meal replacements (IF-P only).

IF days consisted of ~350–550 kcals per day, in which participants were provided a variety of supplements and snacks. Protein pacing (P) days for IF-P consisted of four and five meals/day for women and men, respectively, two of which (breakfast and one other meal) were liquid meal replacement shakes with added whole foods (Whole Blend IsaLean® Shakes, 350/400 kcals, 30/36 g of protein/meal, 9 g of fiber); a whole food evening dinner meal (450/500 kcals men), an afternoon snack (200 kcals, men only), and an evening protein snack (IsaLean® or IsaPro® Shake or IsaLean Whole Blend® Bar; 200–250 kcals). This dietary regimen provided 1350–1500 and 1700–1850 kcals/day for women and men, respectively, and a macronutrient distribution targeting 35% protein, 35% carbohydrate, 20–30 g/day of fiber, and 30% fat. Isagenix International, LLC (Gilbert, AZ, USA) provided all meal replacement shakes, bars, beverages, and supplements. In comparison, participants assigned to the CR diet followed specific guidelines of the National Cholesterol Education Program Therapeutics Lifestyle Changes (TLC) diet of the American Heart Association with a strong Mediterranean diet influence of a variety of fresh vegetables, fruits, nuts, and legumes. The specific macronutrient distribution recommended was <35% of kcal as fat; 50%–60% of kcal as carbohydrates; 15% kcal as protein; <200 mg/dL of dietary cholesterol; and 20–30 g/day of fiber. The total calorie intake was 1200 and 1500 calories per day for women and men, respectively, during the 8-week weight loss intervention. In addition to weekly meetings with the registered dietitian and daily contact with research team members, subjects were provided detailed written instructions for their meal plans. They were closely monitored through daily participant-researcher communication (e.g., email, text, and mobile phone), two-day food diary analysis, weekly dietary intake journal inspections, weekly meal/supplement container distribution, and returning empty packets and containers.

Gastrointestinal (GI) symptom rating scale

Participants completed the 15-question GI symptom rating scale (GSRS) 84 at baseline, week four, and week eight. Briefly, each question is rated on a 7-point Likert scale (1 = absent; 2 = minor; 3 = mild; 4 = moderate; 5 = moderately severe; 6 = severe and 7 = very severe) and recalled from the previous week. Questions include symptoms related to upper abdominal pain, heartburn, regurgitation (acid reflux), empty feeling in the stomach, nausea, abdominal rumbling, bloating, belching, flatulence, and questions on defecation. The GSRS questionnaire provides explanations of each symptom, is understandable, and has reproducibility for measuring the presence of GI symptoms 85 . In our analysis, a score of ≥2 (minor) was defined as symptom presence, and a score ≥ 4 (moderate) was defined as moderate symptom presence. Furthermore, to better categorize symptom location, bloating, flatulence, constipation, diarrhea, stool consistency, defecation urgency, and sensation of not completely emptying bowels were classified as lower GI symptoms, and nausea, heartburn, regurgitation, upper abdominal pain, empty feeling in the stomach, stomach rumbling, and belching was classified as upper GI symptoms. Total scores were also generated for overall symptom and moderate symptom presence.

Fecal sample collection and DNA extraction

Participants were instructed to provide stool samples at baseline, week four, and week eight of the intervention. The case-study participant additionally provided samples at weeks 12, 16, 32, and 52. The entire bowel movement was collected and transported within 24 h of defecation to the Skidmore College Human Nutrition and Metabolism (Saratoga Springs, NY) laboratory using a cooler and ice packs and frozen at −80 °C. Samples were then sent to ASU (Phoenix, AZ) overnight on dry ice for analysis, where they were thawed at 4 °C and processed. Wet weight was recorded to the nearest 0.01 g after subtracting the weight of fecal collection materials. Stool samples were then rated according to the BSS 86 , homogenized in a stomacher bag, and the pH was measured (Symphony SB70P, VWR International, LLC., Radnor, PA, USA). Next, the extraction of DNA was performed using the DNeasy PowerSoil Pro Kit (Cat. No. 47016, Qiagen, Germantown, MD) per the manufacturer’s instructions. DNA concentration and quality were quantified using the NanoDrop™ OneC Microvolume UV-Vis Spectrophotometer (Thermo Scientific™, Waltham, MA) according to manufacturer instructions. The OD 260 /OD 280 ratio of all samples was ≥1.80 (demonstrating DNA purity).

Quantification of bacterial 16S rRNA genes

To estimate total bacterial biomass per sample (16S rRNA gene copies per gram of wet stool), DNA extracted from the fecal collections was assessed via quantitative polymerase chain reaction (qPCR) based on previously published methods 87 , 88 . Briefly, all 20 μL qPCR reactions contained 10 uL of 2X SYBR Premix Ex Taq ™ (Tli RNase H Plus) (Takara Bio USA, Inc., San Jose, CA, USA), 0.3 μM (0.6 μL) of each primer (926 F: AAACTCAAAKGAATTGACGG; 1062 R: CTCACRRCACGAGCTGAC), 2 μL DNA template (or PCR-grade water as negative control), and 6.8 μL nuclease-free water (Thermo Fisher Scientific, Waltham, MA, USA). PCR thermal cycling conditions were as follows: 95 °C for 5 min, followed by 35 cycles of 95 °C for 15 s, 61.5 °C for 15 s, and 72 °C for 20 s, then hold at 72 °C for 5 min, along with a melt curve of 95 °C for 15 s, 60 °C for 1 min, then 95 °C for 1 s. Quantification was performed using a QuantStudio3™ Real-Time PCR System by Applied Biosystems with QuantStudio Design and Analysis Software 1.2 from Thermo Fisher Scientific (Waltham, MA, USA). All samples were analyzed in technical replicates. For quality assurance and quality control, molecular negative template controls (NTC) consisting of PCR-grade water (Invitrogen, Waltham, MA, USA) and positive controls created by linearized plasmids were run on every qPCR plate. Standard curves were run-in triplicate and used for sample quantification, ranging from 10 7 to 10 1 copies/μL with a cycle threshold (CT) detection limit cutoff of 33. Reaction efficiency was approximately 101%, with a slope of −3.29 and R 2  ≥ 0.99.

Fecal microbiome analysis

Amplification of the 16S rRNA gene sequence was completed in triplicate PCRs using 96-well plates. Barcoded universal forward 515 F primers and 806 R reverse primers containing Illumina adapter sequences, which target the highly conserved V4 region, were used to amplify microbial DNA 89 , 90 . PCR, amplicon cleaning, and quantification were performed as previously outlined 90 . Equimolar ratios of amplicons from individual samples were pooled together before sequencing on the Illumina platform (Illumina MiSeq instrument, Illumina, Inc., San Diego, CA). Raw Illumina microbial data were cleaned by removing short and long sequences, sequences with primer mismatches, uncorrectable barcodes, and ambiguous bases using the Quantitative Insights into Microbial Ecology 2 (QIIME2) software, version 2021.8 91 .

16S rRNA sequencing produced 7,366,128 reads with a median of 53,776 per sample (range: 9512–470,848). Paired-end, demultiplexed data were imported and analyzed using QIIME2 software. Upon examination of sequence quality plots, base pairs were trimmed at position 20 and truncated at position 240 and were run through DADA2 to remove low-quality regions and construct a feature table using ASVs. Next, the ASV feature table was passed through the feature-classifier plugin 92 , which was implemented using a naive Bayes machine-learning classifier, pre-trained to discern taxonomy mapped to the latest version of the rRNA database SILVA (138.1; 99% ASVs from 515 F/806 R region of sequences) 93 . Based on an assessment of alpha rarefaction, a threshold of 6500 sequences/sample was established, retaining all samples for downstream analysis. A phylogenic tree was then constructed using the fragment-insertion plugin with SILVA at a p-sampling depth of the rarefaction threshold to impute high-quality reads and normalize for uneven sequencing depth between samples 94 . Alpha diversity (intra-community diversity) was measured using observed ASVs and the Phylogenetic diversity index. Additionally, the Shannon index was calculated for the subgroup and case-study analyses to capture richness and evenness at the species level. Beta diversity (inter-community diversity) was measured using Bray-Curtis dissimilarity.

For shotgun metagenomics, DNA was sequenced on the Illumina NextSeq 500 platform (Illumina, CA, USA) to generate 2 × 150 bp paired-end reads at greater sequencing depth with a minimum of 10 million reads. Raw Illumina sequencing reads underwent standard quality control with FastQC. Adapters were trimmed using TrimGalore. DNA sequences were aligned to Hg38 using bowtie2 95 . DNA sequences were then analyzed via the bio bakery pipeline 96 for taxonomic composition and potential functional content with MetaPhlAn4 and HUMAnN 3.0 (UniRef90 gene-families and MetaCyc metabolic pathways), using standard parameters. Functional profiling resulted in 8528 distinct Kyoto Encyclopedia of Genes and Genomes Orthology (KO) groups and 511 metabolic pathways, which align with previous human gut microbiome studies 96 .

Blood sample collection and biochemical analyses

All participants were tested between the hours of 6:00 a.m. and 9:00 a.m., after an overnight fast for body composition assessments (height, body weight, and total body composition) at weeks 0, 4, and 8. 12-h fasted venous blood samples (~20 mL) were collected into EDTA-coated vacutainer tubes and centrifuged (Hettich Rotina 46R5) for 15 min at 4000 ×  g at −4 °C. After separation, plasma was stored at −80 °C until analyzed. Undiluted plasma samples were sent to Eve Technologies (Calgary, Alberta, Canada) for assessment of inflammatory cytokines [Granulocyte-macrophage colony-stimulating factor [GM-CSF], interferon-γ (IFNγ), interleukin (IL)-β, IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12p70, IL-13, IL-17A, IL-23, and Tumor necrosis factor-α (TNFα)] using a high human sensitivity 14-plex cytokine assay (Millipore, Burlington, MA). Circulating LBP concentrations were quantified in duplicate using 1000x diluted plasma samples. A commercially available kit was used per the manufacturer’s protocol (Cat No. EH297RB, Thermo Fisher Scientific, Inc, Waltham, MA; intra-assay coefficient variation [CV] <10%).

Targeted plasma metabolomic analysis

For the plasma metabolomic analysis, a 12-h fasted venous blood sample (~20 mL) was collected into EDTA-coated vacutainer tubes and centrifuged (Hettich Rotina 46R5) for 15 min at 4000 ×  g at 4 °C. After separation, 2 mL of plasma was aliquoted and stored at −80 °C at the Biochemistry Laboratory at Skidmore College (Saratoga Springs, NY, USA). Samples were then sent to the Arizona Metabolomics Laboratory at ASU (Phoenix, AZ, USA) overnight on dry ice for analysis, where they were thawed at 4 °C and processed. Briefly, 50 μL of plasma from each sample was processed to precipitate proteins and extract metabolites by adding 500 μL MeOH and 50 μL internal standard solution (containing 1810.5 μM 13 C 3 -lactate and 142 μM 13 C 5 -glutamic acid). The mixture was vortexed (10 s) and stored for 30 min at –20 °C, then centrifuged at 224,000 ×  g for 10 min at 4 °C. Supernatants (450 μL) were extracted, transferred to new Eppendorf vials, and dried (CentriVap Concentrator; Labconco, Fort Scott, KS, USA). Samples were then reconstituted in 150 μL of 40% phosphate-buffered saline (PBS)/60% acetonitrile (ACN) and centrifuged again at 22,000 ×  g at 4 °C for 10 min. Supernatants (100 µL) were transferred to an LC autosampler vial for subsequent analysis. Quality control (QC) was performed by creating a pooled sample from all plasma samples and injecting once every ten experimental samples to monitor system performance.

The highly-reproducible targeted LC–MS/MS method used in the current investigation was modeled after previous studies 97 , 98 , 99 . The specific metabolites included in our targeted detection panel are representative of more than 35 biological pathways most essential to biological metabolism and have been successfully leveraged for the sensitive and broad detection of effects related to diet 100 , diseases 101 , drug treatment 102 , environmental contamination 103 , and lifestyle factors 104 . Briefly, LC–MS/MS experiments were performed on an Agilent 1290 UPLC-6490 QQQ-MS system (Santa Clara, CA, USA). Each sample was injected twice for analysis, 10 µL using negative and 4 µL using positive ionization modes. Chromatographic separations were performed in hydrophilic interaction chromatography (HILIC) mode on a Waters Xbridge BEH Amide column (150 × 2.1 mm, 2.5 µm particle size, Waters Corporation, Milford, MA, USA). The flow rate was 0.3 mL/min, the autosampler temperature was maintained at 4 °C, and the column compartment was set at 40 °C. The mobile phase system was composed of Solvents A (10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% H 2 O/5% ACN) and B (10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% ACN/5% H 2 O). After the initial 1 min isocratic elution of 90% Solvent B, the percentage of Solvent B decreased to 40% at t  = 11 min. The composition of Solvent B was maintained at 40% for 4 min ( t  = 15 min).

The mass spectrometer was equipped with an electrospray ionization (ESI) source. Targeted data acquisition was performed in multiple-reaction monitoring (MRM) mode. The LC–MS system was controlled by Agilent MassHunter Workstation software (Santa Clara, CA, USA), and extracted MRM peaks were integrated using Agilent MassHunter Quantitative Data Analysis software (Santa Clara, CA, USA).

GC–MS fecal short-chain fatty acid analysis

Before GC–MS analysis of SCFAs, frozen fecal samples were first thawed overnight under 4 °C. Then, 20 mg of each sample was homogenized with 5 μL hexanoic acid—6,6,6-d 3 (internal standard; 200 µM in H 2 O), 15 μL sodium hydroxide (NaOH [0.5 M]), and 500 μL MeOH. Samples were stored at −20 °C for 20 min and centrifuged at 22,000 ×  g for 10 min afterward. Next, 450 μL of supernatant was collected, and the sample pH was adjusted to 10 by adding 30 μL of NaOH:H 2 O (1:4, v-v). Samples were then dried, and the residues were initially derivatized with 40 µL of 20 mg/mL MeOX solution in pyridine under 60 °C for 90 min. Subsequently, 60 µL of MTBSTFA containing d 27 -mysristic acid was added, and the mixture was incubated at 60 °C for 30 min. The samples were then vortexed for 30 s and centrifuged at 22,000 ×  g for 10 min. Finally, 70 µL of supernatant was collected from each sample and injected into new glass vials for GC–MS analysis.

GC–MS conditions used here were adopted from a previously published protocol 105 . Briefly, GC–MS experiments were performed on an Agilent 7820 A GC-5977B MSD system (Santa Clara, CA); all samples were analyzed by injecting 1 µL of prepared samples. Helium was the carrier gas with a constant flow rate of 1.2 mL/min. Separation of metabolites was achieved using an Agilent HP-5 ms capillary column (30 m × 250 µm × 0.25 µm). Ramping parameters were as follows: column temperature was maintained at 60 °C for 1 min, increased at a rate of 10 °C/min to 325 °C, and then held at this temperature for 10 min. Mass spectral signals were recorded at an m/z range of 50–600, and data extraction was performed using Agilent Quantitative Analysis software. Following peak integration, metabolites were filtered for reliability. Only those with QC CV < 20% and a relative abundance of 1000 in > 80% of samples were retained for statistical analysis.

Untargeted fecal metabolomic analysis

Briefly, each fecal sample (~20 mg) was homogenized in 200 µL MeOH:PBS (4:1, v-v, containing 1810.5 μM 13 C 3 -lactate and 142 μM 13 C 5 -glutamic Acid) in an Eppendorf tube using a Bullet Blender homogenizer (Next Advance, Averill Park, NY). Then 800 µL MeOH:PBS (4:1, v-v, containing 1810.5 μM 13 C 3 -lactate and 142 μM 13 C 5 -glutamic Acid) was added, and after vortexing for 10 s, the samples were stored at −20 °C for 30 min. The samples were then sonicated in an ice bath for 30 min. The samples were centrifuged at 22,000 ×  g for 10 min (4 °C), and 800 µL supernatant was transferred to a new Eppendorf tube. The samples were then dried under vacuum using a CentriVap Concentrator (Labconco, Fort Scott, KS). Prior to MS analysis, the obtained residue was reconstituted in 150 μL 40% PBS/60% ACN. A quality control (QC) sample was pooled from all the study samples.

The untargeted LC–MS metabolomics method used here was modeled after that developed and used in a growing number of studies 106 , 107 , 108 . Briefly, all LC–MS experiments were performed on a Thermo Vanquish UPLC-Exploris 240 Orbitrap MS instrument (Waltham, MA). Each sample was injected twice, 10 µL for analysis using negative ionization mode and 4 µL for analysis using positive ionization mode. Both chromatographic separations were performed in hydrophilic interaction chromatography (HILIC) mode on a Waters XBridge BEH Amide column (150 × 2.1 mm, 2.5 µm particle size, Waters Corporation, Milford, MA). The flow rate was 0.3 mL/min, autosampler temperature was kept at 4 °C, and the column compartment was set at 40 °C. The mobile phase was composed of Solvents A (10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% H 2 O/5% ACN) and B (10 mM ammonium acetate, 10 mM ammonium hydroxide in 95% ACN/5% H 2 O). After the initial 1 min isocratic elution of 90% B, the percentage of Solvent B decreased to 40% at t  = 11 min. The composition of Solvent B maintained at 40% for 4 min ( t  = 15 min), and then the percentage of B gradually went back to 90%, to prepare for the next injection. Using mass spectrometer equipped with an electrospray ionization (ESI) source, we collected untargeted data from 70 to 1050 m/z.

To identify peaks from the MS spectra, we made extensive use of the in-house chemical standards (~600 aqueous metabolites), and in addition, we searched the resulting MS spectra against the HMDB library, Lipidmap database, METLIN database, as well as commercial databases including mzCloud, Metabolika, and ChemSpider. The absolute intensity threshold for the MS data extraction was 1000, and the mass accuracy limit was set to 5 ppm. Identifications and annotations used available data for retention time (RT), exact mass (MS), MS/MS fragmentation pattern, and isotopic pattern. We used the Thermo Compound Discoverer 3.3 software for aqueous metabolomics data processing. The untargeted data were processed by the software for peak picking, alignment, and normalization. To improve rigor, only the signals/peaks with CV < 20% across quality control (QC) pools, and the signals showing up in >80% of all the samples were included for further analysis. To ensure the robustness of our model validation, we employed an enhanced validation approach by repeating the LOOCV process 100 times. Each iteration involves excluding one sample from the dataset to serve as the test set, with the model being trained on the remaining samples. This approach, referred to as ‘repeated LOOCV’, was adopted to mitigate bias and provide a thorough validation of our model’s predictive capability. The method signifies the number of repetitions of the LOOCV process, rather than splitting the dataset into 100 equal parts.

Multi-omics data analysis

For MOFA, bacterial 16S rRNA ASVs and plasma metabolites were integrated using the MOFA2 package 55 . Before integration, ASV sequences were filtered (minimum of 5 ASV in greater than 10% of all samples), collapsed to the genus level, and scaled using a centralized-log-ratio, as described previously 109 . Plasma metabolites were scaled and normalized as described in the metabolome analysis. The inputs for MOFA model training comprised 53 taxa and 138 metabolites. The latent factors and feature loadings were extracted from the best-trained model with the built-in functions of MOFA2. After model fitting, the number of factors was estimated by requiring a minimum of 2% variance explained across all microbiome modalities.

Integrating microbial taxa with the same filtration as stated above (at the genus level from 16S amplicon sequencing and species level from metagenomic sequencing) and cytokine data and fecal metabolomic data, respectively, was conducted with GFLASSO (R package: GFLASSO, v0.0.0.9000). This correlation-based network solution can handle multiple response variables for a given set of predictors (in this case: 1. cytokine abundances predicted by microbial taxa response; and 2. fecal metabolite response predicted by microbial taxa). Solution parsimony was determined by an unweighted (i.e., presence or absence of association by imposing a correlation threshold) network structure. The regularization and fusion parameters were determined from the smallest root mean squared error (RMSE) estimate via cross-validation, accounting for interdependencies among microbial features. The tested parameters encompassed all combinations between λ and γ with values ranging from 0 to 1 (inclusive) in step increments of 0.1. GFLASSO coefficient matrices were constructed using a threshold coefficient of >0.02 to discern the strongest associative signals.

Statistical analysis

Gastrointestinal symptom scores were on the low end of the GSRS scale and not normally distributed; therefore, nonparametric statistical tests were applied. Symptom prevalence (number of scores ≥ 2) and moderate symptom prevalence (≥4) for total, upper, and lower GI GSRS clusters were analyzed using contingency tables. Specifically, differences between IF-P and CR GI symptoms at baseline were compared using a Fisher’s Exact test, whereas baseline vs. weeks four and eight values were compared with McNemar’s test. Stool weight, BSS, fecal pH, plasma cytokines and LBP, and SCFAs were assessed for normality with Q-Q plots and Shapiro-Wilk tests and log-transformed where appropriate. These were then tested for time and interaction (group × time) effects using linear-mixed effect (LME) models, with each participant included as a random effect.

For analysis and visualization of the microbiome data, artifacts generated in QIIME2 were imported into the R environment (v4.2.2) using the phyloseq package (v1.42.0) 110 . Before conducting downstream analyses, sequences were filtered to remove all non-bacterial sequences, including archaea, mitochondria, and chloroplasts. After assessing normality (Shapiro-Wilk’s tests), LME models were used to test the effect of time and the interaction of group and time with the covariates of age and sex with each participant included as a random effect on the alpha diversity metrics using the nLME package (v3.1.160). For beta diversity, a nested permutational analysis of variance (PERMANOVA) was conducted on Bray-Curtis dissimilarities using the Adonis test in the vegan package (v2.6.2) with 999 permutations. The PERMANOVA model incorporated the factors of time, individual, interaction (group × time), and participant (nested factor). A permutation test for homogeneity in multivariate dispersion (PERMDISP) was conducted using the ‘betadisper’ function in the vegan package to compare dispersion. To support the Adonis analysis, intra-individual differences were also compared between groups, as previously described 111 , by calculating the within-subject distance for paired samples (baseline vs. weeks four and eight) and testing for group distances (Wilcoxon rank-sum test). Differential abundance analysis was performed using MaAsLin2 (v1.12.0) 18 . To detect changes in microbial features between groups over time, we built linear-mixed models that include group, time, and their interaction, with age and sex as covariates and the participant as a random factor. Before analysis, raw counts from the ASV table were filtered for any sequence not present five times in at least 30% of all samples. A significant p-value for the product term indicates that changes in microbial features differed over time between groups. The Benjamini–Hochberg (BH) procedure was used to correct for multiple testing at ≤0.10. To assess the correlation between changes in specific taxa and biomarkers over the eight-week intervention, Spearman correlation tests were performed.

Univariate and multivariate analyses of plasma metabolites and metabolic ontology analysis were performed, and results were visualized using the MetaboAnalystR 5.0 112 . Human metabolomic data were mapped to the Kyoto Encyclopedia of Genes and Genomes (KEGG) human pathway library to analyze predicted states 113 . The data were log 10 -transformed, and Pareto scaled to approximate normality before all analyses. A GLM was constructed with age, sex, and time as covariates to determine significantly affected metabolites by group intervention. Levene’s test was performed to detect significant homogeneity. The BH procedure was used to correct for multiple testing at ≤0.10. Fecal metabolomic analysis for the subgroup comparison was performed by assessing logFC values between groups with a Wilcoxon rank-sum test with BH adjustment. For pathway analysis, the impact was calculated using a hypergeometric test, while significance was determined using a test of relative betweenness centrality. Importantly, the BH procedure was not applied to pathway and enzyme enrichment analyses for the subgroup assessment since these analyses involve testing the significance of multiple related hypotheses rather than independent hypotheses, which is too conservative, resulting in false negative results.

For MOFA, latent factors explaining ≥2.0% of model variance from the plasma metabolomic and amplicon microbiome data were used to perform Spearman correlations on anthropometric and nutritional data and compared between IF-P and CR groups using Wilcoxon rank-sum tests. The highest beta coefficients (>0.3) detected from GFLASSO models were further assessed by performing Spearman correlations of select microbial features with the response variables (i.e., cytokines and fecal metabolites). All statistical tests were performed with a significance level of p  < 0.05 and BH correction of p .adj < 0.10. In addition, we present data in this study in accordance with the ‘Strengthening The Organization and Reporting of Microbiome Studies’ (STORMS) guidelines for human microbiome research 114 .

Reporting summary

Further information on research design is available in the  Nature Portfolio Reporting Summary linked to this article.

Data availability

The microbiome sequencing data generated in this study have been deposited in the BioProject Database of National Centre for Biotechnology Information database under accession code PRJNA847971 . The metadata data linking the microbiome sequences with the appropriate sample ID and intervention in this study are provided in Supplementary Data  1 . The processed data are available at https://github.com/Alex-E-Mohr/GM-Remodeling-IF-ProteinPacing-vs-CaloricRestriction .  Source data are provided with this paper.

Code availability

The R code used for analysis and figure generation for reproducibility purposes are available at: https://github.com/Alex-E-Mohr/GM-Remodeling-IF-ProteinPacing-vs-CaloricRestriction . 115

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Acknowledgements

We thank the trial volunteers for their dedication and commitment to the study protocol. We are grateful for the research assistants from Skidmore College who provided valuable assistance with study protocol design, scheduling, recruitment, data testing, collection, entry, and statistical analysis, and preparation of manuscripts: Molly Boyce, Jenny Zhang, Melissa Haas, Olivia Furlong, Emma Valdez, Jessica Centore, Annika Smith, Kaitlyn Judd, Aaliyah Yarde, Katy Ehnstrom, Dakembay Hoyte, Sheriden Beard, Heather Mak, and Monique Dudar. We are grateful for the extensive guidance and counseling provided by the registered dietitian Jaime Martin. We thank research coordinator Michelle Poe for her superior dedication to all aspects of the study. This study was primarily funded by an unrestricted grant from Isagenix International LLC to P.J.A. (grant #:1911-859), with secondary funding provided to K.L.S.

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Contributions

Study conceived and designed: P.J.A. Manuscript preparation with input from all authors: A.E.M., K.L.S., D.A.B., P.J., C.M.W., D.D.S., R.K.-B., H.G., J.K.-S., K.M.A., E.G., and P.J.A. Randomized study design and execution: K.M.A., and P.J.A. Microbiome analysis: A.E.M., D.A.B., C.M.W., and R.K.-B. Blood analyte analysis: A.E.M., K.L.S., and P.J.A. Metabolomic analysis: A.E.M., Y.J., H.G., and P.J. Statistical analysis and data presentation: A.E.M., C.M.W., D.D.S., R.K.-B., and P.J.A. Supervision and funding: K.L.S., E.G., and P.J.A.

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Competing interests.

P.J.A. is a consultant for Isagenix International LLC, the study’s sponsor, he is an advisory board member of the International Protein Board (iPB), and he receives financial compensation for books and keynote presentations on protein pacing ( www.paularciero.com ). Eric Gumpricht is employed by Isagenix International, LLC, the funding source for this research. Isagenix International, LLC had no role in the study design, data collection, analysis, or decision to publish. No authors have financial interests regarding the outcomes of this investigation. The other authors declare no competing interests.

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Mohr, A.E., Sweazea, K.L., Bowes, D.A. et al. Gut microbiome remodeling and metabolomic profile improves in response to protein pacing with intermittent fasting versus continuous caloric restriction. Nat Commun 15 , 4155 (2024). https://doi.org/10.1038/s41467-024-48355-5

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DOI : https://doi.org/10.1038/s41467-024-48355-5

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