PONE-D-22-13220Cocoa intake and muscle pain sensation: a randomized experimental studyPLOS ONE

Dear Dr. Christidis,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewerrìs raised serious critiques to some key methodological aspects and also on other parts of the manuscript. Please assess if you are able to address their concerns appropriately. 

Please submit your revised manuscript by December 29th. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

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Andrea Martinuzzi

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Partly

Reviewer #2: Partly

Reviewer #3: No

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2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: I Don't Know

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3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: *** General comments: ***

The design is a three-period three-treatment crossover design with the further complication of including a two-factor within-subject design with multiple evaluations over time in each period. This design requires special techniques to analyze correctly. See Senn (2002) for details about methodology. The current analysis is incorrect.

The manuscript lacks line numbers, therefore, it is difficult to provide specific suggestions. There are minor grammatic errors that might benefit from a third-party proof-reading.

In addition, it does not seem that the data are available for review.

*** Specific comments: ***

The authors presumably have already found that their design is not going to be handled correctly using SigmaPlot -- however, the repeated measures analysis that is proposed is incorrect. Also, it seems likely that the implied R-side correlation structure implemented by SigmaPlot is too simplistic. Further, the use of the Friedman test is even more limited in its ability to correctly analyze these data.

This crossover design features:

* 3 periods

* 3 treatments

* 6 sequences

* 30 subjects

The within period design features:

* 2 conditions (pre- and post-chocolate)

* Multiple time-points

* Multiple measures

While it is not clear that performing a correct analysis would lead to substantially different results, it is difficult to say ahead of actually performing it. A correct analysis will include treatment, sequence, subject, period, and carryover effects in the between-subject design, and pre/post and time in the within-subject design. It may be reasonable to omit the carryover, but this must be justified. Senn (2002) provides an excellent discussion of this issue that may be of help.

As mentioned in the limitations, there is most likely a period effect. Therefore, this must be accounted in the analysis at a minimum. The randomization restriction represented by the sequence is also important. A subject-level effect is required to produce the correct degrees of freedom for analysis (and potentially, depending on how the analysis is specified, a random effect of subject by period). Most likely, the authors would like to generalize their results past this specific set of subjects, so that subject should be considered as a random effect.

This design should probably be analyzed using either SAS or R. It is recommended to use SAS, unless the authors have access to expertise in using R to specify complex analyses. In SAS it would be feasible to implement the entire design as run using PROC MIXED. However, setting up the analysis does require a little experience, effort, and likely some trial and error and it would require both G-side and R-side specifications.

Having set this analysis up would provide several benefits. First, it allows all tests of interests to be performed as either omnibus tests or contrasts (whether corrected for multiple testing or not). Second, it allows covariates to be entered to evaluate their effects net of the treatment or vice versa. Third, it provides a template that correctly reflects the experiment design in all its complexity and can be used for all assessments (ideally).

Alternatively, while the fine-grained sampling over time is interesting, it seems to provide little benefit in the actual analysis. Therefore, a simpler method of analyzing the data would be as follows:

* Summarize the data across time using a statistic or statistics, e.g.:

- Area under the curve

- Peak value

- Other

* Determine the differential effect of chocolate using the summary statistic, e.g.:

- Post-chocolate minus pre-chocolate

- Ratio of post-chocolate to pre-chocolate

* Perform a standard 3-treatment 3-period crossover analysis

Some sample PROC MIXED code for this "simpler" analysis would be:

PROC MIXED;

CLASS SEQUENCE SUBJECT PERIOD TREATMENT;

MODEL DELTA = TREATMENT PERIOD;

RANDOM SUBJECT(SEQUENCE);

LSMEANS TREATMENT / PDIFF CL E;

RUN;

(Note that the code above could be extended to allow the pre/post x time design within subjects, and this would indeed be the starting point for that effort.)

A side benefit of this approach is that it allows an easier comparison of the various measures with each other via scatterplots or correlation analysis. It is also more likely to provide data that meet the assumptions of the linear model analysis.

The authors state that they evaluate normality of the data. This is a misnomer. One can easily see that in any case of interest with actual group differences that the data will not be normally distributed. What is assessed is the normality of the residuals from the linear model analysis. (Or, as in this case, suitably standardized or studentized residuals.)

Even if these residuals are not completely consistent with normality, it is usually preferable to use the same analysis structure throughout rather than a hodge-podge of statistical methods. Nonparametric analogues can be used as support for this. Recall that linear models still produce unbiased results even if error distributions are not normal. This pays dividends in interpretability and ease of representing the experimental design.

Note that the authors present significance tests based on ranks, but then quantitative results based on means. This is a common method to try to dodge the fact that rank-based methods provide no real quantitative statements but strikes one as disingenuous. Using a linear model approach is better for this purpose, even if the model fit is only fair.

*** Reference: ***

Senn, S. S. (2002). Cross-over trials in clinical research (Vol. 5). John Wiley & Sons.

Reviewer #2: This paper reports on a generally well-designed study producing novel data that intake of chocolate reduces acute pain induced by hypertonic saline injection into the masseter muscle of young healthy men and women. There are however several points that need to be satisfactorily addressed by the authors before it could be considered acceptable.

1. The first 4 paragraphs of the Introduction inexplicably focus on chronic pain, yet this study did not address chronic pain per se since it used an experimental model of acute pain which the Discussion states is used “to mimic clinical acute muscle pain”. The 1st part of the Introduction thus needs to be re-cast with a focus on acute pain and underlying mechanisms, including those expressed in the masticatory muscles.

2. The introduction’ s middle paragraph about cocoa inhibiting “trigeminal nerve cells……spinal cord” needs modification since trigeminal nerve cells predominantly project to the brainstem, not spinal cord.

3. There seems to be a disconnect in the description of the Study Design. Clearer wording is needed. Items in pain scores are said to have been graded on a 0-10 scale but this is followed by the statement that they were graded from 0 to 4.

4. Timelines are given in the Methods text and Flow Chart but It is not convincing that there was sufficient time for chocolate ingestion to have occurred to exert analgesic effects (eg, via modulation of neurogenic inflammation) especially during the initial time points where the pain reductions are extremely modest. Also the time designation on the Time axis in Figs 3 and 4 are tiny and very difficult for the reader with even 20/20 vision to discern.

5. The Study Design outlines measures to assess both pain intensity and pain disability, but the Results overlooks any presentation of the latter.

6. Strengths of the study included the use of different concentration of cocoa in the chocolate pieces ingested, and the finding that taste preference played a key role. The 1st couple of paragraphs of the Discussion discuss cocoa and its analgesic effects but it is unclear how a definitive conclusion could be reached from the present study that it is the cocoa in the ingested pieces of chocolate that was responsible for the modest analgesic effects. There were other ingredients in the chocolate (soy, sugar, vanilla) and there is no indication in the study design that these were controlled for, despite different cocoa concentration being used. Accordingly, the title of the paper should be modified to reflect “chocolate” not ‘cocoa’.

7. The !st sentence of the Discussion does not make sense; it seems to be missing words.

8. Likewise, the 1st sentence of the 9th paragraph about taste preference being “responsible for the response to cold pain tolerance” is unclear and needs to be re-formulated to make it clear how taste preference could affect a “response” to the “tolerance” of “cold pain”. The next 2 sentences also have a problem in the formatting (and show grammatical errors).

9. The Study Limitations will need to include those identified above (eg, Point 4).

Reviewer #3: The subject could be of great interest however, the design and the interpretation of the results are a limitation. The main concern is that the authors do not know or not show the composition of the chocolate. White chocolate has only cocoa fat with no flavanols at all (30% of cocoa fat is a lot really). They omit the sugar content in the different chocolates that should be around 50% for milk and white chocolate. Maybe these facts can better explain the preference and the un-expected results regarding the "higher flavanols the higher analgesic effect". I think that, unfortunately, it is not possible to get to any conclusion because there is no real control for the effect. It looks to me that it is the sugar content the one producing any effect and to probe it you would need a non -sugar content control

When authors say that is a controlled study: Which is the control?

The conclusion “The results indicate that it is not mainly the cocoa-concentration that has a positive effect on pain-characteristics, but the effect seems to be affected by the preference” is not based in the results. There is no significance or correlation to support it. Again I think the difference lays in the sugar content.

I don’t see the point of using an abbreviation for serotonin.

Did the authors check for a sequence effect in the assays (white, milk or dark) on the preference for one or other chocolate?

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6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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PONE-D-22-13220R1Chocolate intake and muscle pain sensation: a randomized experimental studyPLOS ONE

Dear Dr. Christidis,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

The reviewers still highllight some major problems in the statistical analysis that critically undermine the validity of the work. Consider if you are able to address the issue otherwise consider withdrawal and possibly resumbission under different headline or journal.

Please submit your revised manuscript by MArch 30th. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Andrea Martinuzzi

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

Reviewer #4: (No Response)

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Yes

Reviewer #3: Partly

Reviewer #4: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: I Don't Know

Reviewer #4: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: No

Reviewer #4: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have satisfactorily addressed my remaining points from the previous review, except for point 7 since the first sentence still is unclear: how could it be that findings that “the pain reduction by the different types of chocolate presented in this study is line with previous studies “ (11, 48-50); none of these 4 cited studies investigated the effects of chocolate on pain. The sentence seems unnecessary and could be deleted unless the authors modify this sentence to clarify exactly what they mean by this unclear sentence.

Also note the revised wording on line 211 has introduced inappropriate tense changes in the same sentence; past tense should be used for both sets of verbs.

Reviewer #3: I think that the composition of the chocolates, now that it has been included, clearly shows that the effect, if any, is not due to the bioactives in cocoa, polyphenols and methylxanthines, but to maybe the cocoa fat and the sugar. In fact, the results show that in the case of men the sugar and fat content with no cocoa bioactives is more effective than the bioactive containing cocoa of dark chocolate.

I think that all this should be included in the abstract. It does not seem to be a question of preference but a question of sugar (and maybe fat contents)

This is in disagreement with most papers showing an effect of cocoa polyphenols and methylxanthines at different levels

See for instance:

Goya L, Kongor JE, de Pascual-Teresa S. From Cocoa to Chocolate: Effect of Processing on Flavanols and Methylxanthines and Their Mechanisms of Action. Int J Mol Sci. 2022;23(22):14365. Published 2022 Nov 18. doi:10.3390/ijms23221436

or

Gu Y, Lambert JD. Modulation of metabolic syndrome-related inflammation by cocoa. Mol Nutr Food Res. 2013;57(6):948-961. doi:10.1002/mnfr.201200837

etc

However, I think the paper should be published showing the results they got with really low amounts of cocoa solids (0% in white chocolate and only 9% in milk chocolate).

It is very important to show that this assay was done based on the bioactivity of cocoa (polyphenols and methylxanthines) however, the real content of this compounds, that was not determined, should be really low since cocoa fat has not beneficial effects that we knew until now.

Reviewer #4: The paper presents a randomized double-blinded controlled trial. First and foremost, the most important question if this is a powered trial to detect a significant effect. With 30 samples and without any power analysis this is not a powered trial. So any claim of significance based on p-value does not make sense.

Second, I reviewed the previous round of reviews and agree with the statistical reviewer that this should be analyzed using a crossover design. A mixed-effect model with a period effect should be used. The previous reviewer has given some excellent guidelines on how to analyze the data which I agree with. He/she also has SAS code, which was pretty tough. Unfortunately, all the suggestions are ignored for two reasoning. 1. They do not have access to SAS and 2. The authors have used similar simpler methods earlier which are accepted.

This is not a constructive way to address the reviewer’s suggestions. First, if SAS is not available, please use R, which is completely free to use. Second, just because previous papers are not reviewed by a statistician thoroughly it does not mean the methods used by them are all appropriate. It seems the investigative teams lack a statistician as otherwise converting the given SAS code to R and reanalyzing the data using a crossover design is not a big issue. The reviewer all mentions several other challenges that have to do with normality and model fitting which I cannot comment on given the suggestion is ignored.

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7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #2: No

Reviewer #3: No

Reviewer #4: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

While revising your submission, please upload your figure files to the Preflight Analysis and Conversion Engine (PACE) digital diagnostic tool, https://pacev2.apexcovantage.com/. PACE helps ensure that figures meet PLOS requirements. To use PACE, you must first register as a user. Registration is free. Then, login and navigate to the UPLOAD tab, where you will find detailed instructions on how to use the tool. If you encounter any issues or have any questions when using PACE, please email PLOS at figures@plos.org. Please note that Supporting Information files do not need this step.

PONE-D-22-13220R2Chocolate intake and muscle pain sensation: a randomized experimental studyPLOS ONE

Dear Dr. Christidis,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

There are still major ponits to be addressed (see comments of reviewer 3).

Please submit your revised manuscript by April 20th. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. When you're ready to submit your revision, log on to https://www.editorialmanager.com/pone/ and select the 'Submissions Needing Revision' folder to locate your manuscript file.

Please include the following items when submitting your revised manuscript:

• A rebuttal letter that responds to each point raised by the academic editor and reviewer(s). You should upload this letter as a separate file labeled 'Response to Reviewers'.
• A marked-up copy of your manuscript that highlights changes made to the original version. You should upload this as a separate file labeled 'Revised Manuscript with Track Changes'.
• An unmarked version of your revised paper without tracked changes. You should upload this as a separate file labeled 'Manuscript'.

If you would like to make changes to your financial disclosure, please include your updated statement in your cover letter. Guidelines for resubmitting your figure files are available below the reviewer comments at the end of this letter.

If applicable, we recommend that you deposit your laboratory protocols in protocols.io to enhance the reproducibility of your results. Protocols.io assigns your protocol its own identifier (DOI) so that it can be cited independently in the future. For instructions see: https://journals.plos.org/plosone/s/submission-guidelines#loc-laboratory-protocols. Additionally, PLOS ONE offers an option for publishing peer-reviewed Lab Protocol articles, which describe protocols hosted on protocols.io. Read more information on sharing protocols at https://plos.org/protocols?utm_medium=editorial-email&utm_source=authorletters&utm_campaign=protocols.

We look forward to receiving your revised manuscript.

Kind regards,

Andrea Martinuzzi

Academic Editor

PLOS ONE

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: (No Response)

Reviewer #3: (No Response)

Reviewer #5: (No Response)

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2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #2: Partly

Reviewer #3: No

Reviewer #5: Partly

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: I Don't Know

Reviewer #3: Yes

Reviewer #5: No

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #5: No

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #2: Yes

Reviewer #3: Yes

Reviewer #5: No

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #2: The authors have satisfactorily addressed the remaining points in my most recent review. However, I do not believe that the authors have satisfactorily addressed the point of Reviewer #3 (which was also related to a point that I raised in an earlier review) that sugar or other ingredients in the chocolate could have contributed to the reduction in pain and that this should be included in the Abstract. The revised Abstract now in its conclusion only refers to taste preference for sugar to explain the pain reduction, even though now the Discussion (lines 684-685) indicates that “other ingredients in the chocolate that could explain this chocolate-induced reduction of pain variables” and the Conclusion (lines 708-709) states that “Another possible explanation could be the composition of the chocolate, i.e. the concentration of the other ingredients such as sugar, soy, and vanilla. Comparable wording should be briefly reflected in the Abstract conclusion.

Reviewer #3: The abstract keeps being unclear and now I think that intentionally misleading. White chocolate might have 30% cocoa fat, but no flavanols at all (introduction and hypothesis is based on this). The results clearly show that if any the pleasure of fat plus sugar intake (that is what white chocolate and milk chocolate mainly provide) is the one producing the "analgesic" effect, very interestingly and in a statistically significant way in the case of white chocolate in men. I think that this needs to be said from the abstract.

Reviewer #5: The authors, in this round of revision, did their best to address the concerns raised. However, they relegated the suggestion of conducting a full-scale crossover design. It is not clear why they were reluctant to conduct it. I am OK to pass the paper, but more arguments are needed behind not using a cross-over design as a separate analysis.

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Reviewer #2: No

Reviewer #3: No

Reviewer #5: No

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Chocolate intake and muscle pain sensation: a randomized experimental study

PONE-D-22-13220R3

Dear Dr. Christidis,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

Reviewer #5: All comments have been addressed

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Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #5: (No Response)

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3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #5: (No Response)

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Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #5: (No Response)

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Reviewer #2: Yes

Reviewer #3: (No Response)

Reviewer #5: (No Response)

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6. Review Comments to the Author

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Reviewer #2: (No Response)

Reviewer #3: I thank you the authors for clarifying partially the real conclusion in this study. It is their paper and if they prefer to keep the reader in the confusion that flavanols might have something to do with any of this I won't ask for rejection. However, and this is my personal recommendation, I think that the authors should know and let the readers know that there should be 0 flavanols in white chocolate, the cocoa % correspond to cocoa fat and there are no flavanols in cocoa fat. Additionally in the methodology they could give an idea of the flavanol content expected in these kind of product, that should be 0 for white chocolate and really low for the milk chocolate. I think that talking about cocoa content when the content is of cocoa fat is not rigorous.

In my opinion, in fact, the design of the study (the product chosen for the study) is not correct for the hypothesis. Maybe the authors should say so as a limitation of their study.

Reviewer #5: (No Response)

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Reviewer #2: No

Reviewer #3: No

Reviewer #5: No

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