Dear Dr. Basualto-Alarcón,

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.

MAJOR DETAILS:

1. The structure of the manuscript, especially the METHODS section, needs to be revised. All subsections should be organized in strict order as recommended by CONSORT guidelines for pilot and feasibility studies (Trial design, Participants, Intervention Procedures, Outcomes, Sample Size, Statistical Analysis) as informed in the submission guidelines of PLoS One. Cf, PMID 31608150 or www.equator-network.org.

Was the clinical trial registered? Platforms such as ClinicalTrials.gov, OSF, or Figshare are available for this purpose.

Were participants aware that they were receiving the creatine monohydrate supplement (i.e., open-label study), or were they blinded to the intervention (i.e., single-blind)? Consider including this information in the title for greater clarity.

This study is not a prospective observational study. This is a single-arm repeated-measures feasibility clinical trial (revise Line 126 and line 300). This is an experimental study.

The authors should clarify which variables are considered primary outcomes and which are secondary outcomes.

Was dietary intake or physical activity recorded during the study?

Please provide additional details regarding the blood sampling procedures, the test protocols used to evaluate biomarkers, and the equipment or devices employed in the laboratory.

Have the formulas been validated in the study population, or at least in a Latin American population? If not, this should be acknowledged as a limitation. 

2. Replace conventional bioelectrical impedance analysis with bioelectrical impedance vector analysis (BIVA). If you used the mBC 565 device, you should present BIVA results—such as the Xc versus R plot normalized by stature, or even specific BIVA using available body girth measurements—instead of relying solely on body composition estimates of SMM, ICW, FFM, ECW, and TBW. While phase angle is a valuable absolute outcome of BIVA, it can be further complemented with indices like the BI index (Height²/Z at 50 kHz) and the impedance ratio (Z at high and low frequencies in kHz).

"An IR ratio closer to 1 is indicative of cell membrane disruption, allowing more fluids, proteins, and electrolytes to shift into the extracellular space [22]. A strong inverse correlation has been reported between the phase angle (PhA) and IR in different clinical populations [22]. PhA is defined as the delay in current flow caused by a reduction in cell membrane capacitance [25]." Cf, PMID: 38488531

3. Statistical analysis. 

This section requires revision. Due to the small sample size and the fact that the required statistical power was not achieved (i.e., fewer participants than indicated by the sample size calculation), the authors should avoid null hypothesis significance testing (NHST). Instead, the analysis should focus on estimation methods—reporting 95% confidence intervals and unbiased effect sizes (e.g., Cohen's d, also known as Hedges' g)—as well as robust statistics, such as trimmed means and Winsorized standard deviations. Please re-run the analysis accordingly and confirm whether the findings and conclusions remain consistent. Thanks to your open data sharing, I have attached the raw data Excel file along with an example of the results output from Jamovi, which you may use for organizing and presenting your findings.

Cf,

- Estimation statistics: https://pubmed.ncbi.nlm.nih.gov/24220629/

"ESCI" module in Jamovi

- Robust statistics: https://pubmed.ncbi.nlm.nih.gov/31152384/

"Walrus" module in Jamovi

In addition, please generate and replace current figures with estimation plots to display the repeated measures data across time points (at baseline and after creatine supplementation). Cumming or Gardner-Altman estimation plots are recommended (see examples of these figures in the attached Excel file).

Please add the CV% of the instruments/technician to know the variability of all measures.

MINOR DETAILS: 

- Please adjust the background and hypothesis in the introduction secion (as well as in the abstract) to creatine related information. The introduction can be reduced to one page only. Missing key references on this topic: 

https://pubmed.ncbi.nlm.nih.gov/31083291/

https://pubmed.ncbi.nlm.nih.gov/28110688/

https://pubmed.ncbi.nlm.nih.gov/34444869/

- Although "body weight" and "height" are frequently used terms, it is technically correct to refer to "body mass" and "stature", respectively. Please address this accordingly throughout the manuscript.

- Dynamometer strength measurements should be expressed in kilogram-force (kgF). Please revise accordingly and replace "Strength" with "Handgrip Strength" in Figure 1. 

- Do not use "mean±standard deviation." Use Mean(SD) instead. Cf, PMID 21206631

- The results of all variables should be expressed as Δ ± SD [95% CI of the change]; unbiased Cohen's [with the corresponding 95% CI]. Remember that unbiased Cohen's = Hedge's g

- Please edit the tables to present all relevant findings, including effect sizes, 95% confidence intervals, and other pertinent statistics.

- Line 374: The authors should highlight that this is one of the first clinical trials to evaluate the effects of oral creatine monohydrate supplementation on BIVA variables, strength-related outcomes, and functional capacity in hemodialysis patients. 

- The discussion should place greater emphasis on BIVA-related data (e.g., bioimpedance indices reflecting cellular integrity), strength-related outcomes, and estimates of body composition (e.g., skeletal muscle mass, fat-free mass, etc.).

- The discussion on intra- and extra-celullar water might be enriched if the impedance ratio is reported (see comments above).

- In agreement with the reviewers, the authors should elaborate on potential confounding variables (e.g., diet, physical activity, season time, etc.) that could have influenced or biased the findings. 

- Additional information that should be referenced in the discussion to support the safety of creatine supplementation on renal health includes: 

https://pubmed.ncbi.nlm.nih.gov/31375416/

https://pubmed.ncbi.nlm.nih.gov/38869518/

https://pubmed.ncbi.nlm.nih.gov/38874125/ 

- Please provide a short paragraph of limitations before conclusions.

- Acknowledge conflicts of interest (if any) in the acknowledgements section.

- Replace the "STROBE checklist" with "CONSORT for feasibility and pilot studies checklist"

==============================

Please submit your revised manuscript by Jun 06 2025 11:59PM. 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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We look forward to receiving your revised manuscript.

Kind regards,

Prof. Diego A. Bonilla

Academic Editor

PLOS ONE

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Additional Editor Comments:

Dear authors,

The manuscript "Oral creatine in hemodialysis patients increases physical functional capacity and muscle mass" is interesting and could be a valuable contribution to the literature.

This clinical study aimed to determine whether oral creatine monohydrate supplementation improves muscle mass and physical functional performance in a convenience sample of adult dialysis patients, with measurements taken before and after supplementation. However, the authors need to address the reviewers' concerns and revise the manuscript in accordance with the editor's comments to improve its structure and data analysis, thereby enhancing its scientific robustness. I commend the authors for supporting open science and sharing their data.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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

The PLOS Data policy

Reviewer #1: Yes

Reviewer #2: No

Reviewer #3: Yes

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: Peer Review: "Oral Creatine in Hemodialysis Patients Increases Physical Functional Capacity and Muscle Mass"

Below is my peer review of the manuscript entitled "Oral Creatine in Hemodialysis Patients Increases Physical Functional Capacity and Muscle Mass." This study is very interesting and timely. However, the authors need to address some of my concerns prior to acceptance for publication.

Major Concerns:

1. Absence of a Control Group

• Description: The study employs a single-arm, before-and-after design without a control group. All 18 participants received creatine supplementation (5 g/day for 8 weeks), and outcomes were compared pre- and post-intervention without a comparator group receiving placebo or standard care.

• Reference:

• Methods section, "Patient Selection" (Page 14, Lines 124-137): "All the recruited patients received creatine supplementation. They were measured before (pre-) and after (post-) receiving creatine supplementation."

• Introduction (Page 13, Line 118): It is an observational study.

• Impact: The lack of a control group is a significant methodological flaw that compromises the study's internal validity. Without a control, it is impossible to attribute the observed improvements in skeletal muscle mass, intracellular water, phase angle, and Short Physical Performance Battery (SPPB) scores solely to creatine supplementation. Confounding factors such as the placebo effect, regression to the mean, natural recovery, seasonal variations (e.g., vitamin D levels increased from winter to summer, Page 21, Lines 241-245), or increased attention from researchers could explain the results. This limitation undermines the study's ability to establish causality, a critical aspect for a research article claiming that creatine "increases" functional capacity and muscle mass, as stated in the title and conclusion (Page 11, Lines 60-63).

• Significance: For a pilot study, a single-arm design may be exploratory, but the absence of a control group severely limits the strength of the conclusions, especially given the definitive language used (e.g., "improved muscular and functional outcomes," Page 11, Line 61).

• Recommendation: Revise the manuscript to explicitly acknowledge the lack of a control group as a significant limitation in the Discussion section (beyond the brief mention on Page 28, Line 400). Temper the conclusions to reflect the preliminary nature of the findings, e.g., change "increases physical functional capacity and muscle mass" (Title, Page 1) to "may increase" or "is associated with increases in." Suggest a randomized controlled trial (RCT) as the next step to confirm causality.

• Rationale: This adjustment aligns the interpretation with the study's observational design and mitigates overstatement, preserving credibility.

2. Insufficient Sample Size

• Description: The study calculated a required sample size of 25 patients based on an effect size of 0.6, alpha of 0.05, and power of 0.8 for detecting changes in SPPB scores (Page 17, Lines 182-190). However, only 19 patients were recruited, and 18 completed the study, falling short of the planned sample size.

• Reference:

• Methods section, "Sample size calculation" (Page 17, Lines 182-190): "The required sample size... is 25 patients... we were only able to approach the calculated sample size."

• Results section, "Baseline Characteristics" (Page 19, Lines 210-216): "We were initially able to recruit only 19... From this initial selection, we had to withdraw one patient."

• Impact: Failing to meet the calculated sample size raises concerns about the study's statistical power. Although significant results were reported (e.g., SPPB p=0.043, skeletal muscle mass p=0.009, Page 22, Table 2), an underpowered study increases the risk of type II errors (missing true effects) and may inflate the risk of type I errors (false positives), particularly with borderline p-values like 0.043 for SPPB.

• Significance: The small sample size, combined with the lack of a control group, amplifies doubts about the robustness of the conclusions. The authors acknowledge this limitation (Page 28, Line 399), but the shortfall still constitutes a significant concern given the study's reliance on statistical significance to support its claims.

• Recommendation: Expand the Discussion to detail the implications of the reduced sample size on statistical power and result reliability (beyond the current note on Page 28, Line 399). Report post-hoc power analyses for key outcomes (e.g., SPPB, skeletal muscle mass) to clarify the strength of the findings. Recommend future studies with adequate sample sizes based on the observed effect sizes (e.g., 0.53 for SPPB, Page 25, Line 335).

• Rationale: Transparency about power limitations strengthens the manuscript's integrity and guides future research design.

3. Selection Bias Due to Convenience Sampling

• Description: The study used a convenience sample of patients selected based on "good adherence to pharmacological and dialysis therapy" (Page 14, Lines 125-126). This non-random selection method resulted in a cohort that may not represent the broader hemodialysis population.

• Reference:

• Methods section, "Patient Selection" (Page 14, Lines 124-131): "A convenience sampling... primarily based on their good adherence to pharmacological and dialysis therapy."

• Results section, "Baseline Characteristics" (Page 19, Lines 210-216): Describes recruitment challenges, noting exclusions due to mobility issues and poor adherence.

• Impact: Selecting patients with good adherence introduces selection bias, as these individuals may be healthier, more compliant, or more motivated than the average hemodialysis patient. This bias limits the external validity of the findings, as the results may not generalize to the broader population, particularly those with poorer adherence or more severe conditions who might respond differently to creatine supplementation. The manuscript does not adequately address how this selection impacts the applicability of the findings to clinical practice.

• Significance: This methodological flaw questions the study's relevance to the target population stated in the introduction (Page 13, Lines 88-93), where hemodialysis patients are described as a vulnerable group with high morbidity and mortality. The biased sample weakens the claim that creatine could mitigate these outcomes across all hemodialysis patients (Page 11, Lines 61-63).

• Recommendation: Discuss the potential impact of selection bias in the Discussion section, noting that patients with good adherence may differ from the general hemodialysis population in health status or behavior. Adjust claims about generalizability (e.g., Page 11, Lines 61-63) to specify the study population (e.g., "in adherent hemodialysis patients"). Propose stratified sampling or broader inclusion criteria in future studies.

• Rationale: Acknowledging and contextualizing this bias enhances the manuscript's applicability and prevents overgeneralization.

Conclusion

The manuscript presents valuable preliminary data suggesting that creatine supplementation may benefit hemodialysis patients by improving muscle mass and functional capacity. However, the identified significant concerns—lack of a control group, insufficient sample size, and selection bias—fundamentally undermine the study's ability to establish causality, reliability, and generalizability. These issues do not invalidate the research entirely, especially given its pilot study context, but they require substantial revision to ensure scientific soundness.

Additional Recommendation for Future Research

Consider a double-blind RCT with a placebo control group, a more prominent and more representative sample, and monitoring of confounders (e.g., physical activity, diet) to address all three concerns comprehensively.

Reviewer #2: This study evaluated the effect of 8 weeks of daily creatine monohydrate supplementation in patients with end stage CRF on dialysis.

The study reported improvements in SPPB and ICW with an increase in FFM and muscle mass.

Comments to consider:

1. The study was open label and there was no control group. As the authors indicated, most subjects started in the winter and finished in the summer. This was likely one contributor to the higher vitamin D (in addition to the 8 who were supplemented) BUT was also likely associated with more physical activity. Most healthy people and patients tend to do more physical activity in the summer vs winter and this could confound the SPPB scores. The trend for those who had increased (I am assuming that the levels increased over time and not increased above normal) was rather strong (P = 0.085) suggesting that vitamin D could be a factor (keep in mind that the sample size was small for the sub-group).

2. I do not see that the authors instructed the patients to control for diet, hydration status and pre-measurement exercise that can confound the BIA measurements.

3. The lack of change in body weight and a ~ 2 kg increase in FFM implies that body fat was down ~ 2 kg - the authors need to comment on this and provide some explanation.

4. The authors should discuss the limitations of the BIA and the lack of another method for the analysis such as DEXA.

Reviewer #3: This is a very interesting study. The hypothesis is well founded and based on relevant literature and the methods and results are sound.

In my opinion, this study should certainly be accepted for publication, provided some minor revisions are made.

The primarily limitation is the open label nature of the study, but this issue can be resolved by sufficiently stating this.

Comments:

Comment 1: Please include a short mention in the methods what an increase in phase angle represents.

Comment 2: The open label nature of the study should be reflected in the abstract. Please include the words “open label” or state there was no control group.

Comment 3: The term protein-energy-wasting is not mentioned in the introduction or discussion, please incorporate this too.

Comment 4: “Different nutritional supplements have also been explored, However, its implementation is often associated with higher costs and the need for trained personnel to ensure its proper operation.”, please elaborate which supplements are referred to with this statement, or omit alltogether.

Comment 5: The quality of the figures appears to be quite low. Please improve to 300 dpi.

Comment 6: How was intracellular water calculated? Please add.

Comment 7: How was missing data handled in the analyses?

Comment 8: Please define how dry weight was determined.

Comment 9: Please report in SI units, i.e. μmol/L rather than mg/dL.

Comment 10: There is no mentioning of side effects in the results section (only shortly in the discussion). Please report on this in the results section too. Did patients experience for instance bloating/nausea or interdialytic weight gain due to the extra volume?

Comment 12: Do you have any explanation why no effect was seen on handgrip strenght, although this was hypothesized? Please discuss this.

Comment 13: Line 86, omit additional space; Line 100, double end of sentence point after [8-9]

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Attachments

Attachment

Submitted filename: Raw Project Data -Creatine renal.xlsx

Oral creatine in hemodialysis patients increases physical functional capacity and muscle mass, an open label study

PONE-D-25-15109R1

Dear Dr. Basualto-Alarcón,

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.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

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Kind regards,

Prof. Diego A. Bonilla

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Dear Authors,

Thank you for addressing the revisions to enhance the manuscript's scientific robustness and transparency.

To further strengthen the paper, please consider these final suggestions:

- In "Data Analysis Procedures", add a brief statement clarifying how the robust analytical approach mitigated the limitations of not reaching the a priori sample size target.

- Revise some references in the discussion:

RE, "Kim HJ, et al. Amino Acids. 2011;40:1409–18." with "Kreider RB, et al. J Int Soc Sports Nutr. 2025;22(sup1):2488937. doi:10.1080/15502783.2025.2488937, PMID 40198156"

In line 338: Highlight creatine’s role as a conditionally essential nutrient across the lifespan, citing: Kreider RB, et al. Front Nutr. 2025;12:1578564. doi:10.3389/fnut.2025.1578564, PMID 40331098 as well as the already cited paper by Post A, Tsikas D, Bakker SJL (2019).

Reviewers' comments: