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Thank you for the opportunity to improve our manuscript. The following sentences are in lines 147-149 in the original manuscript and have been added to the title page in the revised manuscript (lines 28-29). “This clinical trial has been registered with clinicaltrials.gov (Identifier: NCT006672497). Data collection started on July 26, 2021 and is ongoing through May 1, 2025.” The additional sentence, “The authors confirm that all ongoing and related trials for this observation trial (no drug or intervention) are registered.” has been added to the title page and in lines 152-153 of the revised manuscript.

(Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: The aims of the study are to develop a protocol that 1) assesses mitochondrial dysfunction in cancer patients using exercise that is tolerable for the patients throughout the entire chemotherapy regimen and 2) explores the link between oxygen utilization at the muscular level via NIRS with whole body traditional metabolic ventilatory measures.

It seems to be a two-step process. First the investigators will use an observational mixed model repeated measures design to longitudinally compare (pre-chemotherapy and throughout treatment) patients’ skeletal muscle mitochondrial capacity of the vastus lateralis during stationary cycling. The random effect for slope and intercept are reasonable. However, some of the detail is not quite clear. What is the rationale for an unstructured correlation?

Many thanks to the reviewer for comments to improve our manuscript. Based on the work of Cheng et al. [35], they suggest that with a “fixed effect of time (chemotherapy cycle), “time may be included as a categorical predictor in the fixed and random effects, which corresponds exactly to a fully saturated polynomial design matrix in time. In such cases, a wide range of covariance structures has appeal, especially unstructured patterns. Similar to the selection of fixed effects, the evaluation of covariance structure should depend on scientific knowledge in the specific area as well as statistical criteria.” Since this is the first study to measure mitochondrial function during chemotherapy, there is little to no evidence to support another approach. See other references (33-36 for further support. We have added two sentences to elaborate on this rationale per the reviewer’s comments in line (171-172).

Also, the eligibility criteria has to be clarified a bit more for model purposes. Will breast cancer and gynecologic subjects be analyzed separately? What specifically would be the ineligibility criteria?

Thank you for the comments to improve our manuscript. Because the treatment for the gynecological patients and the breast cancer patients is very similar per NCCN guidelines, both populations will be analyzed together. In reality, they are all women and all adjuvant. At the time of administration, there is no clinical evidence of disease (non-metastatic). Therefore, the patients that come in are similar from a population viewpoint. The regimens have much the same overlap. It’s disease “agnostic”. The drugs/treatment regimens are much the same. If the treatment is significantly different based on their oncologist’s recommendation, then the populations would be analyzed separately. We have added 2 sentences (lines 192-193) to clarify the manuscript.

The independent variables are therapy and number of treatments. There are three dependent variables which will increase the number of analyses.

Also, how will the covariates listed on the left side of Figures 1A and 1B be incorporated into the analysis? Will this be a repeated ANCOVA instead of an ANOVA?

Thank you for the comments to improve our manuscript. In figure 1A and 1B, the additional outcome measures are being collected as additional whole-body vs. muscle level cardio-metabolic health. With 30 subjects, we do not anticipate having enough data points to sub-divide the data by these measures to have meaningful analysis. Meaning, the statistical assumptions would not be able to be met. This additional data could be considered more holistically in a clinical case study. If warranted, another future study could collect more data to statistically analyze with these as true covariates.

What accommodation will be made in the sample size of just 30 for dropouts or missing data?

Thank you for the comments to improve our manuscript. We have added a sentence (lines 180-182) stating that data collection will continue until we reach 30 subjects who have completed the study, excluding dropouts or subjects whose data was unusable due to missing data.

One has difficulty anticipating how the analysis will be organized and reported for this endeavor? When the investigators say that, ‘We assume that at least n=8 of each chemotherapy type will be necessary to detect any differences in trajectory of τ over time’, what does that mean exactly? How many chemotherapy types are there exactly for these diagnoses?

Thank you for the comments and opportunity to improve the manuscript. We have revised lines 182-191 and added additional sentences to reflect what we have seen in pilot data so far and in conversations with several of the oncologist in the Cancer Institute. Based on the NCCN guidelines, treatment can be considered cytotoxic (platinum based, anthracyclines, etc.) or non-cytotoxic such as immunotherapy. These treatments can be given separately or at the same time. Generally, they consist of 3 or more cycles. Therefore, we have added revisions to reflect that differences between cytotoxic and non-cytotoxic treatment could be considered based on current NCCN guidelines. If the NCCN guidelines change based on research or development of new drug regimens, comparisons, could also change.

The second step is obviously the oxygenation via NIRS which the authors explain in a separate data analysis and statistical analysis section. The organization of the presentation is challenging to navigate through. The authors perhaps should re-edit this paper more directed to presenting the material in a protocol topic format .

Thank you for the comments to improve our manuscript. We have revisited the PLOS One Study Protocol Article Template to make sure we are following the proper format. We have edited the methods as well as added several sentences throughout for clarification and to improve readability.

Reviewer #2: This study has a well-designed protocol with clear goals to assess the impact of chemotherapy on mitochondrial function in cancer patients. The experimental design, equipment, and statistical analysis are appropriate for the research objectives. However, several points need to be clarified before being considered for publication:

1) I question the necessity of explicitly stating that the results will be shared through conferences and publications in the last sentence of the abstract. But this made me wonder if your results are likely not going to be published ? If so this should be addressed in the manuscript for transparency.

Thank you for the comments to improve our manuscript. We have every intention of publishing our results in peer-reviewed journals in the field. We have added the word “journal” to the last sentence of the abstract (line 59 revised manuscript) and line 406 in the last sentence of the discussion.

Since “Data collection started on July 26, 2021 and is ongoing through May 1, 2025.” but only 13 out of the 30 planned participants have been enrolled so far, it seems unlikely the enrollment goal will be reached.

Thank you for the comments to improve our manuscript. We are on track for meeting our goal of 30 patients and have completed testing of 26 out of 30 patients at the time of these revisions. This change has been reflected in line 583 of the revised manuscript. It is very common with patient recruitment to have a slower rate than other normal human subject studies. Patient recruitment with clinical trials, especially with cancer patients can sometimes be as low as 2-4 patients per year. We have extended the end date of the IRB for recruitment to March 27, 2026 and reflected this in revising line 152.

2) Weight loss or cachexia status is not explicitly mentioned as a tracked variable. Not all cancer patients and not all patients undergoing chemotherapy develop cachexia. Some may maintain weight, while others experience severe muscle wasting and cachexia itself affects mitochondrial function, independent of chemotherapy, potentially confounding the study’s main outcome. The manuscript needs to mention this and separate analyses for cachectic vs. non-cachectic patients should be ran to determine whether cachexia modifies the effect of chemotherapy on mitochondrial function.

Thank you for this observation and comments to improve our manuscript. As a pilot observational study, our primary outcome measure is reduction in mitochondrial function of patients going through chemotherapy. The mechanism of why there is mitochondrial dysfunction is multi-factorial including effects from cachexia, but it is not our intention to elucidate this underlying mechanism, only the outcome of reduced mitochondrial capacity during chemotherapy. Chemotherapy induced mitochondrial dysfunction has a more rapid effect vs. the slower effect of cachexia. Our primary measurements do not include the measurement of cachexia. However, we do understand that the wording in the manuscript may confuse readers, therefore have improved wording that streamlines our “if/then” algorithm. While chemotherapy can induce cachexia, it does have a component of severe inflammatory skeletal muscle response. Much of this is due to mitochondrial dysfunction. That being said, we agree with the reviewer that the patient may or not have cachexia and have clarified wording in the manuscript concerning cachexia in lines 85-93.

3) The introduction contains becomes difficult to follow due to several flow and grammatical issues, or incomplete sentences and erroneous statements. The entire manuscript should be carefully reviewed for proper language.

Thank you for this comment. Introduction has been edited to reflect this change.

For example:

- cachexia and chemotherapy-induced muscle wasting are related but distinct conditions and should not be distinguished (line79: “chemotherapy-induced muscle wasting, also known as cachexia”).

Thank you for this comment. Line 79 has been edited to reflect this change.

- Line 84-86 : "therefore, the increase in survivorship has been an increase in focus on understanding mechanisms of cachexia, such as mitochondrial dysfunction, with the aim of studying these mechanisms to understand how to improve quality of life for [10]."

Thank you for this comment. Lines 84-86 have been edited to reflect this change.

- Line 88-90 “Muscle biopsies have been the gold standard assessment but cause significant discomfort to the patient and ma y not represent in vivo activation of the skeletal working muscle tissue associated with [17].”

Thank you for this comment. Lines 88-90 have been edited to reflect this change.

- Line 109-110: “assessment of age-related differences in skeletal muscle oxidative [17].”

Thank you for this comment. Lines 109-110 have been edited to reflect this change.

- Line 236-239 : “Pictures of the anatomical location of NIRS device placement with a tape measure from the center of the anterior border of the patella to the top of the NIRS device will be taken to standardize placement at every visit.”

Thank you for this comment. Lines 236-239 (lines 250, 259-260 in revised manuscript) have been edited to reflect this change.

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Reviewer #1: No

Reviewer #2: No

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Response:

The authors thank the reviewers for their constructive comments.

We have addressed or commented on all of the points presented, and have compiled a summary of the changes and responses below for reconsideration. The original review text is in pink, and our replies are in black.

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Submitted filename: Respone to Reviewers.docx