Pilot randomized trial of the effect of antibacterial mouthwash on muscle contractile function in healthy young adults

Edgar J Gallardo; William S Zoughaib; Ahaan Singhal; Richard L Hoffman; Andrew R Coggan

doi:10.1371/journal.pone.0312961

. 2025 Feb 12;20(2):e0312961. doi: 10.1371/journal.pone.0312961

Pilot randomized trial of the effect of antibacterial mouthwash on muscle contractile function in healthy young adults

Edgar J Gallardo ^1,^#, William S Zoughaib ^1,^#, Ahaan Singhal ², Richard L Hoffman ¹, Andrew R Coggan ^1,^3,^*

Editor: Ajaya Bhattarai⁴

PMCID: PMC11819566 PMID: 39937800

Abstract

Antiseptic mouthwash use is widespread due to its oral health benefits. However, its impact on systemic physiological processes, particularly nitric oxide (NO) bioavailability and muscle contractility, is not fully understood. We sought to determine the effects of cetylpyridinium (antibacterial) versus sodium chloride (control) mouthwashes on salivary and breath NO markers and muscle contractile function in healthy young adults. Thirty participants (n = 15/group) completed a randomized, parallel-arm, blinded trial, comparing the two mouthwashes before and after 7 d of treatment. NO bioavailability was inferred via measurement of salivary nitrate (NO₃⁻), nitrite (NO₂⁻), and cyclic guanyl monophosphate (cGMP) concentrations and breath NO level. Contractile function of the knee extensor muscles was determined via isokinetic dynamometry. No changes in salivary NO₃⁻, NO₂⁻, or cGMP or in breath NO were observed in response to either treatment. However, cetylpyridinium mouthwash reduced the percentage of NO₂⁻ in saliva (17 ± 10% vs. 25 ± 13%; p = 0.0036). Peak torque at velocities of 0–6.28 rad/s was unaffected by mouthwash use. Calculated maximal knee extensor velocity (Vmax) and power (Pmax) were therefore also unchanged. Cetylpyridinium mouthwash reduces the relative abundance of NO₂⁻ in the oral cavity but does not significantly diminish overall NO bioavailability or impair muscle contractile function in healthy young adults.

Introduction

Approximately $7B worth of antiseptic/antibacterial mouthwash is sold globally each year [1], with roughly ⅓ to ½ of adults in many countries using such products on a regular basis [2–4]. Mouthwash use can diminish plaque formation and reduce gingivitis [5], but such products may also have detrimental effects. Specifically, strong antibacterial mouthwash can disrupt the nitrate (NO₃⁻) -reducing bacteria normally found in the mouth [6–9]. These symbionts reduce salivary NO₃^- to nitrite (NO₂⁻), which after being absorbed can be further reduced to form nitric oxide (NO) [10,11]. NO is a key signaling molecule involved in a variety of physiological responses, including, but not limited to, the regulation of blood flow/pressure. As a result, interruption of this enterosalivary pathway of NO production via twice-daily use of chlorhexidine-containing mouthwash for 3–7 d has been reported to decrease plasma NO₂⁻ levels by 15–25% and increase systolic blood pressure by 2–3 mmHg [12,13]. The latter change may be clinically significant, as it could increase risk of death from ischemic heart disease and stroke by 7% and 10%, respectively [14]. In fact, frequent use of mouthwash has been associated with the development of hypertension [15] and pre-diabetes/diabetes [16]. Chlorhexidine-containing mouthwash has also been shown to block post-exercise reductions in blood pressure [17]. Chronic mouthwash use therefore may have unintended negative consequences.

In addition to influencing blood flow/pressure, NO is involved in the regulation of numerous other physiological functions, including skeletal muscle contractile function [18]. In this regard, numerous studies in recent years have demonstrated that acute ingestion of NO₃⁻-rich beetroot juice can enhance muscle contractility in a wide variety of individuals [cf. Ref. [19] for review]. Although the exact mechanism responsible is still unclear, this improvement has been hypothesized to result from NO-mediated changes in calcium [Ca²⁺] release and/or sensitivity within muscle [20]. Indeed, muscle function seem to be more sensitive to alterations in NO availability than blood pressure, as dietary NO₃^- supplementation has been routinely observed to enhance the former even in the absence of changes in the latter [21–24]. It is not known, however, whether a reduction in NO bioavailability due to use of antibacterial mouthwash can inhibit muscle function. Such knowledge is potentially relevant because muscle strength, speed, and especially power are important determinants of both athletic performance [25] and the ability to perform normal activities of daily living [26].

The purpose of the present parallel-arm study was therefore to determine the effects of antibacterial mouthwash on muscle contractile function in healthy young adults.

Methods

Participants

Potential participants were recruited via word-of-mouth and flyers placed around the university campus. Exclusion criteria were age < 18 or > 30 y; current use of mouthwash, antibiotics, or tobacco products; resting blood pressure > 140/90; an answer of yes to any of the questions of the Physical Activity Readiness Questionnaire (PAR-Q); or inability to provide informed consent. All other persons were included in the study. Data collection took place in two phases, with n = 6 participants studied between November 1^st 2019 and March 13^th 2020 and n = 24 between November 5^th 2020 and March 20^th 2023. All participants studied during the second phase were required to submit to weekly COVID-19 mitigation testing and then later to be vaccinated as a condition of continued university enrollment. Each participant provided written, informed consent, and the study protocol was approved by the Human Subjects Office at Indiana University (protocol number 1907204336). The study was registered at ClinicalTrials.gov (NCT04095442). Participant flow through the study is shown in Fig 1.

Experimental design and protocol

After the initial visit to the Exercise Physiology Laboratory at Indiana University Indianapolis, which included completion of the Physical Activity Readiness Questionnaire (PAR-Q) and practicing the isokinetic dynamometer testing protocol described below, participants were randomized in blocks of four into one of two groups. The randomization scheme was generated using randomization.com by an individual (RLH) not involved in participant recruitment, testing, or data analysis, with the key concealed until completion of the study. One group was studied immediately before and after using Cepacol® mouthwash (Reckitt Benckiser, Parsippany, NJ) for 7 d. This product was chosen as the strongest non-prescription antibacterial mouthwash available in the United States [27]. As a comparison/control, a second group was studied immediately before and after using Tom’s of Maine® Sea Salt Natural Mouthwash (Kennebunk, ME) for 7 d. This product was not expected to have any significant effects on the oral microbiota and hence NO production [28]. These two groups will henceforth be referred to by the active ingredients of the two products, i.e., cetylpyridinium and sodium chloride (NaCl), respectively. The physical characteristics of the participants in each group are shown in Table 1. Twenty-six (87%) of the participants were non-Hispanic White, three (10%) were Black or African American, and one (3%) was Hispanic or Latino, reflective of the racial and ethnic makeup of the local population.

Table 1. Participant characteristics.

	Group
	NaCl	Cetylpyridinium
Total N	15	15
Non-Hispanic White/Black or African American/Hispanic or Latino	14/1/0	12/2/1
Sex (M/F)	8/7	9/6
Age (y)	22 ± 3	23 ± 4
Height (m)	1.74 ± 0.13	1.71 ± 0.05
Mass (kg)	80.1 ± 15.2	76.6 ± 11.3
BMI (kg/m²)	26.5 ± 3.7	26.1 ± 3.3

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Values are means ± SD.

Participants in both groups were instructed to refrain from exercising and to avoid alcohol, caffeine, chewing gum, and foods high in NO₃⁻ (e.g., spinach, beets) for 24 h prior to study visits. Upon reporting to the laboratory, the participant’s breath NO level, a sensitive indicator of changes in whole-body NO production [29], was first measured using a portable electrochemical analyzer (NIOX VERO®, Circassia Pharmaceuticals, Mooresville, NC). A stimulated saliva sample was then collected using a commercial collection kit (Salivette, Sarstedt, Newton, NC). (Stimulated saliva was collected because it is more reflective of plasma NO₃⁻ and NO₂⁻ levels than unstimulated saliva [30].) Due to pandemic-related research restrictions, we were unable to obtain blood samples.) Participants briefly chewed on a synthetic swab until it was saturated with saliva. The swab was then immediately centrifuged for 2 min at 1000 g and 4° C and the saliva frozen at −20 °C until subsequently analyzed for NO₃⁻ and NO₂⁻ concentrations via high performance liquid chromatography (ENO-30, Amuza, San Diego, CA). Salivary cCGMP levels (which are highly correlated with plasma concentrations [31]) were also determined in these samples using a commercial enzyme-linked immunosorbent assay kit (Item number 581021, Cayman Chemical, Ann Arbor, MI). The coefficient of variation for these triplicate measurements averaged 12.0 ± 9.8%.

The contractile properties of the quadriceps muscle group were next determined using an isokinetic dynamometer (Biodex System 4 Pro, Biodex Medical Systems, Shirley, NY) as previously described [21–24]. Briefly, participants performed three maximal knee extensions at angular velocities of 0, 1.57, 3.14, 4.71, and 6.28 rad/s, with 2 min of rest allowed between each set of contractions. The highest torque generated at each velocity was used to calculate peak power at that velocity, after which the maximal velocity (Vmax) and power (Pmax) of knee extension were determined by fitting a parabolic function to the data.

Upon completion of the baseline visit, participants were provided with a bottle of their assigned mouthwash in an opaque bag (to conceal the assignment from the investigators). They were instructed to rinse their mouth as directed on the product’s packaging for 30 s twice per day (i.e., morning and evening, including on the morning of study) for 7 d and to record usage of the mouthwash on a provided form. They were also instructed to maintain their normal oral hygiene habits (e.g., toothbrushing, flossing) but to not use any other mouthwash products during this period. Note that although the two products differed significantly in terms of packaging, color, taste, etc., the participants were kept unaware of the other (i.e., non-assigned) product and were simply told that the purpose of the study was to compare two different mouthwashes, with no expectation as to the outcome. After 7 d, they returned to the laboratory whereupon the above measurements were repeated.

Statistical analyses

Statistical analyses were performed using GraphPad Prism version 10.2.3 (GraphPad Software, La Jolla, CA). Normality of data distribution was tested using the D’Agostino-Pearson omnibus test, whereas Grubb’s test was used to check for possible outliers. Salivary NO₃⁻, NO₂⁻, and log-normalized cGMP concentrations, breath NO levels, and Vmax and Pmax were analyzed using two-way repeated measures analysis of variance (ANOVA), with treatment as a between-subject factor and time as a within-subject factor. Isometric and isokinetic knee extensor peak torque data were analyzed using a three-way repeated measures ANOVA, with treatment as a between-subject factor while velocity and time were treated as within-subject factors. Post-hoc testing was performed using the Holm-Šidák multiple comparison procedure. A two-tailed P < 0.05 was considered significant. Pmax was defined as the primary outcome; all other outcomes were secondary. As this study was the first of its kind, we lacked the preliminary data necessary to perform a formal sample size/power analysis. However, our chosen sample size should have allowed us to detect any change in Pmax with an effect size (i.e., Cohen’s f [32]) ≥ 0.15 with an alpha of 0.05 and 1-beta of 0.95. According to Cohen [32], this would be between a “small” (f = 0.10) and a “medium” (f = 0.25) effect size.

Results and discussion

All eligible participants completed the intervention, with no adverse events in either the NaCl or cetylpyridinium groups. In particular, none experienced a COVID-19 infection during their participation, none reported that they were suffering from long COVID, and none reported any reason why they should not perform physical activity (PAR-Q question 7). However, saliva was available for analysis in only n = 13/group (n = 11 for cGMP in the cetylpyridinium group), whereas due to equipment obsolescence breath NO was only measured in n = 8/group. No other data were missing.

Markers of NO bioavailability

No significant changes were observed in salivary NO₃⁻ or NO₂⁻ concentrations or in their sum in either group (Figs 2A-C). There were also no significant changes in salivary cGMP concentrations or in breath NO levels (Figs 2E, 2F). There was, however, a significant reduction in the relative abundance of NO₂⁻ in the cetylpyridinium group, which decreased from 25 ± 13% to 17 ± 10% of the total salivary NO₃⁻ plus NO₂⁻ concentration (t = 3.512, DF = 24, multiplicity adjusted P = 0.0036) (Fig 2D). The largest decline (i.e., from 54% to 20%) was observed in the individual with the highest initial value. Although not a statistical outlier, the data were nonetheless also analyzed excluding these results. The reduction in salivary NO₂⁻ in the other 12 participants (i.e., from 22 ± 11% to 17 ± 11%) was still highly significant (t = 3.245, DF = 23, multiplicity adjusted P = 0.0071).

Muscle contractile function

Although there was a highly significant effect of velocity on knee extensor peak torque, the effects of time and treatment were not significant, nor were there any significant interaction effects (Table 2). There were therefore no significant changes in either Vmax (Fig 3A) (time: F_1,28 = 0.0007483, P = 0.9784; treatment: F_1,28 = 2.059, P = 0.1624; time x treatment: F_1,28 = 0.4950, P = 0.4875) or Pmax (Fig 3B) (time: F_1,28 = 0.03467, P = 0.8536; treatment: F_1,28 = 0.6123, P = 0.4405; time x treatment: F_1,28 = 0.9878, P = 0.3288).

Table 2. Effect of NaCl- or cetylpyridinium-containing mouthwash on the maximal isometric/isokinetic torque of the knee extensors.

	Group
	NaCl		Cetylpyridinium
Velocity[rad/s]	Pre	Post	Pre	Post	ANOVA results
0	2.65 ± 0.53	2.74 ± 0.55	2.51 ± 0.81	2.46 ± 0.82	Velocity: F_1.607,45 = 125.5, P < 0.0001 Time: F_1,28 = 0.2189; P = 0.6247 Treatment: F_1,28 = 0.1416; P = 0.7095 Velocity x Group: F_4,122 = 0.5770, P = 0.6799 Velocity x Time: F_1.731,48.46 = 0.661, P = 0.4975 Group x Time: F_1,28 = 2.785, P = 0.1063 Velocity x Group x Time: F_4,112 = 0.5019, P = 0.7344
1.57	2.05 ± 0.51	2.14 ± 0.51	2.09 ± 0.66	2.05 ± 0.63
3.14	1.68 ± 0.46	1.70 ± 0.41	1.70 ± 0.59	1.61 ± 0.57
4.71	1.35 ± 0.40	1.37 ± 0.46	1.34 ± 0.48	1.33 ± 0.47
6.28	1.07 ± 0.39	1.13 ± 0.45	1.06 ± 0.45	1.10 ± 0.40

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Values are means ± SD in Nm/kg.

The purpose of this investigation was to determine whether use of over-the-counter antibacterial mouthwash would reduce NO production via the enterosalivary pathway and thus impair muscle contractility in healthy young adults. Contrary to this hypothesis, we found no significant changes in salivary NO₃⁻, NO₂⁻, or cGMP concentrations, in breath NO levels, or in the maximal voluntary isometric/isokinetic torque or speed or power of knee extension. This is the first study to specifically examine the effects of mouthwash use on muscle contractile function or, to our knowledge, any determinant of human exercise performance.

There are several possible explanations for these negative findings. The first is simply that the participants failed to utilize the provided mouthwash as instructed. However, review of the participants’ logs indicated excellent compliance, averaging 97 ± 7% and 97 ± 4% in the cetylpyridinium and NaCl groups, respectively. Furthermore, the relative abundance of NO₂⁻ in saliva was significantly reduced in the cetylpyridinium group, as discussed in greater detail below. Thus, while participant non-compliance cannot be completely excluded as a factor, this explanation seems unlikely.

The second possibility is that twice-daily use of an over-the-counter antibacterial mouthwash for 7 d failed to alter the oral microbiome sufficiently to have any “downstream” consequences. This could be because the bacteria found in the oral cavity are extremely resilient, rapidly repopulating themselves after disruption via physical (i.e., toothbrushing) or chemical (i.e., mouthwash) means [33]. Nonetheless, cetylpyridinium-containing mouthwashes have been shown to suppress salivary bacterial counts by 40–70% for up to 6 h [27] and are highly effective at suppressing growth of and/or eradicating bacteria from biofilms ex vivo [27,33–36]. Cetylpyridinium-based mouthwash has also been found to markedly blunt increases in salivary and plasma NO₂⁻ following an oral NO₃⁻ load [37]. Consistent with these prior observations, in the present study there was a significant (t = 3.512, DF = 24, multiplicity adjusted P = 0.0036) reduction in the percentage of NO₂⁻ in saliva after 7 d of treatment in the cetylpyridinium group. However, there were no changes in the absolute concentrations of either NO₂⁻ or NO₃⁻, suggesting that the effects of cetylpyrdinium on the oral microbiome were somewhat limited.

The third possibility is that cetylpyridinium-containing mouthwash did in fact markedly inhibit NO production via the enterosalivary pathway, but any reduced contribution from this source was simply too small to matter. Consider the following: in the US, normal dietary NO₃⁻ intake is 0.5–1.5 mmol/d [38,39]. However, it has been estimated that only approximately 5% of ingested NO₃⁻ is converted to NO₂⁻ [38–41]. Even assuming that all of this newly-formed NO₂⁻ is ultimately reduced to NO, this implies a maximum rate of NO formation from ingested NO₃⁻ of 0.5–1.5 x 0.05 = 0.025–0.075 mmol/d. At the same time, however, in healthy individuals NOS-mediated NO synthesis is approximately 1 mmol/d [42]. It is therefore clear that the enterosalivary pathway is normally a relatively minor source of NO, a conclusion supported by previous isotopic tracer research [43,44]. As such, use of antibacterial mouthwash might not be expected to have any marked biological effects unless combined with/compared to ingestion of large amounts of NO₃^- [37,45,46] (see below).

As stated previously, to our knowledge this is the first study to determine the influence of mouthwash use per se on muscle contractility or any other aspect of physical function/exercise capacity. A handful of previous investigations, however, have examined the effects of chlorhexidine-containing mouthwash (which is prescription-only in the US, Canada, and some other countries), sometimes in conjunction with a NO₃⁻-restricted diet, on other physiological parameters, particularly blood pressure [8,9,12,13]. Compared to the present results, these studies have generally found much larger changes in salivary NO₂⁻ and NO₃⁻ concentrations, averaging −69% and +80%, respectively, presumably reflecting the more prolonged antibacterial action of chlorhexidine vs. cetylpyridinium [27,33,36]. However, salivary and plasma concentrations of NO₂⁻ are only weakly correlated [8], and changes in plasma NO₂⁻ concentration in response to chlorhexidine use have been much more modest (i.e., −19% on average) [8,9,12,13,47]. Furthermore, no changes have been found in plasma cGMP levels [9,13], a highly sensitive indicator of whole-body NO bioavailability [29,48]. Blood pressure has also been mostly unchanged [8,9,47], with only Kapil et al. [12] and Bondonno et al. [13] reporting small, but statistically significant, increases in systolic blood pressure and only Kapil et al. [13] finding a statistically significant increase in diastolic blood pressure. Again, this could be due to the quantitatively minor contribution of the enterosalivary pathway to NO production under normal circumstances, i.e., at typical levels of dietary NO₃^- intake and in the absence of chronic diseases such as hypertension or heart failure, in which NOS-mediated NO synthesis is reduced [cf. Ref. [42] for review].

There are limitations to the present study. As implied above, we might have obtained different results had we utilized a mouthwash containing chlorhexidine instead of cetylpyridinium, had the participants rinse their mouths more frequently than the recommended two times per day, or had tested the effects of mouthwash in conjunction with a low NO₃⁻ diet or following NO₃⁻ ingestion. Our results might also have been different had we examined some other exercise outcome, e.g., endurance performance ability, instead of muscle contractile function, and/or studied a population in whom NO bioavailability is already reduced (e.g., older individuals). Although salivary [49] (and plasma [50] and urinary [51]) NO₃⁻ and NO₂⁻ levels do not exhibit any diurnal variation, muscle power does [52], such that our findings might still have been different if we had tested at different times of day. We also did not determine the aerobic fitness or body composition of our participants, and cannot rule out an impact of long COVID on their responses. Finally, we only measured the concentrations of NO₃⁻, NO₂⁻, and cGMP in saliva, such that the impact of the intervention on the concentrations of these metabolites in plasma (or more importantly, muscle) is unknown. Inclusion of such measurements, however, would not have changed the fact that our primary outcome, i.e., muscle function, was unaltered. Likewise, quantification of the effects of antibacterial mouthwash on oral NO₃⁻ reducing capacity [53] – which, notably, are not predictive of changes in plasma NO₂⁻ levels [54] - would not have altered our other measured outcomes or their interpretation.

Conclusions

This is the first study to specifically determine the effects of antibacterial mouthwash on muscle contractile function. Although mouthwash use significantly reduced the relative abundance of NO₂⁻ in saliva, consistent with inhibition of the enterosalivary pathway, there were no changes in the concentrations of NO₃⁻, NO₂⁻, and cGMP in saliva and the level of NO in breath, or in maximal muscular strength, speed, or power. Short-term use of cetylpyridinium-based mouthwash does not seem to impair whole-body NO bioavailability or muscle contractility in healthy young individuals.

Supporting information

S1 Data. Statistical analyses.

(DOCX)

pone.0312961.s001.docx^{(154KB, docx)}

S2 Data. Mouthwash study data (final).

(XLSX)

pone.0312961.s002.xlsx^{(76.3KB, xlsx)}

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This study was supported by the Diversity Scholars Research Program of the Center for Research and Learning at Indiana University Indianapolis awarded to EJG. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. 2025 Feb 12;20(2):e0312961. doi: 10.1371/journal.pone.0312961.r001

Author response to Decision Letter 0

16 Aug 2024

PLoS One. doi: 10.1371/journal.pone.0312961.r002

Decision Letter 0

Ajaya Bhattarai

28 Aug 2024

PONE-D-24-32002Randomized trial of the effect of antibacterial mouthwash on muscle contractile function in healthy young adultsPLOS ONE

Dear Dr. Coggan,

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PLOS ONE

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Additional Editor Comments:

The minor revision is needed.

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

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Reviewer #1: Yes

Reviewer #2: Partly

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

Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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Reviewer #1: Yes

Reviewer #2: Yes

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5. Review Comments to the Author

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Reviewer #1: General Comments: The authors have provided a well written manuscript focused on investigating the effect of mouthwash on oral biomarkers of nitric oxide bioavailability and lower extremity skeletal muscle function. Although the question is of relevance given the increasing interest in nitrate supplementation, the population for testing their hypothesis is questionable. Given the support provided in the introduction and the exclusionary criteria of several of the author's manuscripts, it would seem that young healthy adults would not benefit from nitrate supplementation, possibly due to the robustness of physiological reserves at that developmental stage. Thus, the blunting of the enterosalivary pathway with mouthwash would need to have large effect to even expect a reduction in skeletal muscle function in this population. As the authors allude in the discussion, studying this effect in older adults or patient populations that have documented poor NO bioavailability would seem to be the ideal population, especially given emerging evidence of links between oral health and frailty. Nonetheless, in accordance with PLOS ONE publication criteria, the study was conducted well, and I only have minor edits about the framing of the results, questionable statistical analyses, and the preliminary nature of the study.

Specific:

Title Page: This should be labelled a pilot randomized trial for several reasons: 1) No sample size calculation was performed and the effect size from the meta-analysis includes older adults, who we would expect to have a large effect; 2) only 13/15 provided saliva, no blood NO biomarkers, and only 8/15 provided breath NO; 3) The one significant result is potentially driven by an outlier (well discuss further in methods).

Abstract: The authors state "Cetylpyridinium mouthwash INHIBITS reduction of nitrate to nitrite...". This was not tested in this study. The mouthwash was associated with a reduction in salivary NO precursors. Please edit accordingly.

- Keywords should not duplicate words used in the title. Please revise.

Methods:

- Inclusion of participants during the COVID 19 pandemic would necessitate record of if they had previously been exposed and potientially controlling for any differences. Long covid impacts are still being tested but some studies suggest initial NO altering effects [PMID: 37657532]. Please add this as a limitation of your study.

Table 1: Please add Race and Age data. Minorites reports higher exposure to societal stressors that could impact endogenous NO bioavailability. Also, baseline fitness level and body composition were not considered. Given the limitation of BMI and small sample size, differences in aerobic capacity, lean mass, and adipose could impact NO responses. If data is not available, please add as a limitation.

Statistical analyses - a sensitivity analysis should be conducted on data provided in figure D. The reader should know if removal of that one individual in the Cetylpyridinium group with the highest pre value changes the statistical significance of the result. Secondly, the effect size cited should be labeled (i.e., is it Cohen's d, partial eta, etc.) and the reader should know why it was selected.

Results: The focus on percent change is a bit misleading when the individual data points seem to show that one data point is driving the huge change. Please accompany this with the sensitivity analysis data.

Discussion: Given the limitations the findings should be toned down. Please remove any mention of these results being generalizable to older adults or patient populations. Further, exercise performance was not tested. A knee extension is more of a functional outcome. Please use contractility as that is what was tested. Lastly, please add the limitation that one time point was tested (static measure) or provide data that salivary NO or muscle function is not under the influence of circadian biology. IF it is under the influence of circadian biology than it is necessary that the reader is made aware that any significant percentage change is outside the normal within or between day fluctuation. Given that salivary cortisol does fluctuate that does bring some concern as to how to interpret a static salivary measure.

Reviewer #2: This study investigated the effect of antibacterial mouthwash on muscle contractile function in heathy young adults. The manuscript is well written whilst reviewer has some questions.

1) What is the significance of examining this study in young adults? In previous studies, the effects of NO3- supplementation on muscle function have been observed in elderly people with impaired endothelial function and people with vascular disease.

2) It is questionable whether the methods used in this experiment were appropriate. In this study, no increase in NO activity was obtained with the use of mouthwash. This study is considered to be an inappropriate experimental design, as it would only be valid if the use of mouthwash increases NO activity.

3) How reliable is the measurement of breath NO level for assessing NO activity?

4) Mouthwash was used twice a day, but at what time of the day was it used? Also, was the timing of use standardized for all subjects?

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Reviewer #1: No

Reviewer #2: No

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PLoS One. 2025 Feb 12;20(2):e0312961. doi: 10.1371/journal.pone.0312961.r003

Author response to Decision Letter 1

7 Oct 2024

We thank the reviewer for their comments on our manuscript. However, we disagree with their assertion that we tested our hypothesis in the wrong population.

First, although we have published a number of studies of dietary nitrate supplementation in older men and women and patients with heart failure, we have also previously reported that this intervention increases neuromuscular function in healthy young and middle aged untrained men and women (PMCID: PMC4362985) as well as in young athletes (PMCID: PMC4889556). In a follow-up study, we found no apparent effect of age on the magnitude of this effect (PMCID: PMC5789728). This conclusion was supported by our later independent-participant data meta-analysis of the literature (PMCID: PMC8501726), in which the vast majority of participants (i.e., 230 out of 268, or 86%) were young and healthy. It is therefore clear that, contrary to the reviewer’s assertion, dietary nitrate supplementation is beneficial even in individuals such as those included in the present study.

Second, and more importantly, the purpose of the present study was actually to examine the converse question, i.e., to determine whether over-the-counter mouthwash reduced NO levels, and if so, whether this would diminish muscle function. Logically, those most likely to be impacted would be those with normal/adequate levels in the first place – those in whom NO production is already reduced (e.g., older individuals) would presumably be less likely to be impacted (“floor effect”). As it turned out, cetylpyrdinium-based mouthwash seemed to have only a slight influence on salivary markers of NO bioavailability, but this could not have been predicted a priori, as no previous study had tested the effects of cetylpyridinium-containing mouthwash in isolation, only in combination with nitrate supplementation (Ref. 34).

Specific:

Per the reviewer’s suggestion, the title of the manuscript has been changed to “Pilot randomized trial of the effect of antibacterial mouthwash on muscle contractile function in healthy young adults”

The sentence in question has been changed to read “Cetylpyridinium mouthwash reduces the relative abundance of NO2- in the oral cavity . . .”

- Keywords should not duplicate words used in the title. Please revise.

Keywords have been revised.

Methods:

Data collection for out study took place in two phases, with n=6 participants studied between November 1st 2019 and March 13th 2020 and n=24 between November 5th 2020 and March 20th 2023. All participants studied during the second phase were required to submit to weekly COVID-19 mitigation testing and then later to be vaccinated as a condition of continued university enrollment. None of the participants experienced a COVID-19 infection during the study, none reported that they were suffering from long COVID, and none reported any reason why they should not perform physical activity (PAR-Q question 7). This information has been added to the manuscript. Nonetheless, we have added the possible impact of COVID-19 as a limitation to the Discussion.

Racial and ethnic characteristics and mean age of the participants have been added to the Methods section. Lack of detailed information on aerobic fitness and body composition have been added to the Discussion as an additional limitation.

To determine whether our findings were being driven by the results for that single individual, we further analyzed the data two ways:

1) A two-tailed Grubb’s test at P=0.05 was used to test the data for outliers. None were found.

2) The two-way ANOVA was repeated excluding that individual’s results. The decrease in % nitrite in the cetylpyridinium group (i.e., from 22±11% to 17±11%) was still significant (i.e., P=0.0071).

The above information has been added to the manuscript.

Secondly, the effect size cited should be labeled (i.e., is it Cohen's d, partial eta, etc.) and the reader should know why it was selected.

The effect size of 0.15 is actually Cohen’s f statistic, which is the appropriate choice for ANOVA. This is now stated in the manuscript and Cohen’s classic textbook cited. To avoid confusing readers by “mixing and matching” different effect size statistics, we have removed reference to the effect size (Hedge’s g) from our meta-analysis.

Please see above.

Per the reviewer’s suggestions, we have removed any reference to the generalizability of our results from the Discussion, and refer to muscle contractility only as a determinant of exercise performance and/or physical function. We have also added references re. circadian changes (or lack thereof) in our outcome measures, and added the fact that we only tested at one time point as a limitation.

Reviewer #2: This study investigated the effect of antibacterial mouthwash on muscle contractile function in heathy young adults. The manuscript is well written whilst reviewer has some questions.

We thank the reviewer for their compliments re. our writing.

Please see response to reviewer 1.

The reviewer appears to be confused. As stated in the original manuscript, the purpose of our study was to determine whether over-the-counter mouthwash would reduce (not increase, as twice suggested by the reviewer above) NO bioavailability, and if so, whether this had any impact on muscle contractile function. Obviously inhibiting the enterosalivary pathway of NO production via mouthwash use would not be expected to increase NO levels.

3) How reliable is the measurement of breath NO level for assessing NO activity?

Breath NO levels are a well-established and sensitive indicator of changes in whole-body NO production. A reference has been added to this effect.

4) Mouthwash was used twice a day, but at what time of the day was it used? Also, was the timing of use standardized for all subjects?

Participants were instructed to follow the mouthwash manufacturers’ recommendations, which were to use the product morning and evening. This detail has been added to the revised manuscript. Aside from verifying AM and PM use, we did not record the exact timing. As originally stated, however, the effects of cetylpyridinium persist for at least 6 h, which would seem to make the exact timing relatively unimportant.

Attachment

Submitted filename: Response to Reviewers.docx

pone.0312961.s004.docx^{(21.7KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0312961.r004

Decision Letter 1

Ajaya Bhattarai

1 Nov 2024

PONE-D-24-32002R1Pilot randomized trial of the effect of antibacterial mouthwash on muscle contractile function in healthy young adultsPLOS ONE

Dear Dr. Coggan,

Please submit your revised manuscript by Dec 16 2024 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.

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'.

We look forward to receiving your revised manuscript.

Kind regards,

Ajaya Bhattarai

Academic Editor

PLOS ONE

Journal Requirements:

Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Revision is needed on the revised manuscript.

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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 #1: All comments have been addressed

Reviewer #2: All comments have been addressed

Reviewer #3: (No Response)

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

Reviewer #1: Yes

Reviewer #2: Partly

Reviewer #3: Partly

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: No

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

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

Reviewer #1: Yes

Reviewer #2: Yes

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #1: This reviewer is appreciative of the author's responses and believe this manuscript less confusing in its current state. There are only 2 new issues with the manuscript that should be address prior to publication. Well done.

1) As it currently stands, although the overall values are representative of the local population, it is unclear how the 4 minorities are dispersed across the experimental groups. Please add racial/ethnic data and group sample size to table 1 and perform a statistical group comparison and add appropriate p-values to table 1.

2) This study does not provide conclusive data to make this claim in line the final paragraph of the manuscript: "Healthy young Athletes seemingly need not be concerned about possible impairments in muscle contractility due to use

of cetylpyridinium-based mouthwash.". Please remove or add terms of uncertainty.

Reviewer #2: Revised manuscript and the responses to comments were reviewed. I found them to be generally satisfactory.

Reviewer #3: This is a quite an interesting study. However, the manuscript could benefit from further improvements based on the following comments:

Page 4: Apart from the exclusion criteria, inclusion criteria is to be provided.

Page 4: The study design name is to be clearly stated.

Page 5 Paragraph 1 Line 4: The name abbreviation could be attached to the 'individual' as a form of identification.

Pahe 5: The baseline, intervention and post time period is to be clearly stated.

Page 5 Paragraph 1 Line 2: The cut off/requirement or level of acceptance PAR-Q and isokinetic dynamometer is to be stated. The university name is to be stated.

Page 5 Table 1: The table presentation to follow journal format. The decimal point is to be standardized. Title is too short.

Page 7: ‘the latter after log normalization’ is to be rephrased.

Page 7 Paragraph 2 Line 4 & 6: two-way analysis of variance (ANOVA) and three-way analysis of variance (ANOVA) are to be revised to two-way repeated measure analysis of variance (ANOVA) and three-way repeated measure analysis of variance (ANOVA) respectively. The detail analysis in table form is to be presented e.g. main effects, interactions, effect size, post hoc comparison etc and attached as supplementary table. The statistical test assumptions fulfilment is to be stated.

Page 7, Paragraph 2, Line 7: The sentence ‘with treatment as a between-subject factor and velocity and time as within-subject factors’ can be revised to ‘with treatment as a between-subject factor while velocity and time are treated as within-subject factors.

Page 7 Paragraph 2 Line 8: One or two-tailed test for p value is to be stated.

Page 7: The statement ‘According to Cohen [32], this would reflect a “moderate” effect size’ unclear and requires further elaboration. Based on Cohen’s guidelines, Cohen f = 0.15 is considered small and 0.25 considered moderate effect size. 0.15 -0.25 considered small to moderate effect size.

Page 7, Paragraph 2: Missing data (if any) is to be reported. If there is no missing data, a statement indicating this is to be included.

Page 8 Paragraph 2 Line 5: Indicate clearly the p value in Figure 2D.

Page 8 Paragraph 3 Line 1-3 Muscle contractile function: The results (e.g. p values) are to be clearly presented in the table. For the statement ‘nor were there any significant interaction effects (Table 2)’, the results are to be presented.

Table 2: for the table footnote ‘No significant treatment effects were observed’, the p value (s) is/are to be stated/presented. Statistical test is to be denoted in the table footnote. More detailed information such as mean difference, effect size index, p values, 95%CI etc could be presented.

**********

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.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

**********

PLoS One. 2025 Feb 12;20(2):e0312961. doi: 10.1371/journal.pone.0312961.r005

Author response to Decision Letter 2

15 Nov 2024

Table 1 has been expanded to provide the information and statistical comparisons requested.

The offending sentence has been replaced with “Short-term use of cetylpyridinium-based mouthwash does not seem to impair whole-body NO bioavailability or muscle contractility in healthy young Individuals.” Note that this is essentially just a restatement of the results.

Reviewer #3: This is a quite an interesting study. However, the manuscript could benefit from further improvements based on the following comments:

Page 4: Apart from the exclusion criteria, inclusion criteria is to be provided.

As succinctly stated in the original manuscript, “All other persons were included in the study.” We believe that this already makes it clear that anyone between 18 and 30 y of age, who was not currently using mouthwash, antibiotics, or tobacco products, who had a resting blood pressure > 140/90, who answered no to all of the questions of the Physical Activity Readiness Questionnaire (PAR-Q), and was able to provide informed consent was eligible for inclusion. No changes have therefore been made.

Page 4: The study design name is to be clearly stated.

That a parallel-arm design was used is now explicitly stated in the last sentence of the Introduction.

Page 5 Paragraph 1 Line 4: The name abbreviation could be attached to the 'individual' as a form of identification.

The initials of the individual responsible for generating the treatment assignment have been added.

Pahe 5: The baseline, intervention and post time period is to be clearly stated.

It is unclear what the reviewer expects, as this was an acute study, with the intervention period (i.e., 7 d) already clearly stated. Nonetheless, in response we have added the word “immediately” to indicate that there was no delay between the baseline testing and treatment period, or between the treatment period and post-testing.

Page 5 Paragraph 1 Line 2: The cut off/requirement or level of acceptance PAR-Q and isokinetic dynamometer is to be stated. The university name is to be stated.

The name of the university was already explicitly stated just two sentences above, but in response to the reviewer’s comment has also been added here. However, it is completely unclear what they mean by “the cut off/requirement or level of acceptance PAR-Q and isokinetic dynamometer “, so no other changes have been made.

Page 5 Table 1: The table presentation to follow journal format. The decimal point is to be standardized. Title is too short.

We have read carefully the guidelines for table formatting (https://journals.plos.org/plosone/s/tables) and believe that our tables are in conformance. No changes have therefore been made.

Page 7: ‘the latter after log normalization’ is to be rephrased.

This sentence has been revised to read “Salivary NO3-, NO2-, and log-normalized cGMP concentrations . . .”

Please see below.

Both of these comments actually refer to the same two sentences describing the statistical analysis of the data. We believe the fact that repeated measures ANOVAs were performed was already clearly apparent from the nature of the study design and the fact that velocity and time were treated as within-subject factors. Nonetheless, in response to the reviewer’s comment we have now added the words “repeated measures” to both sentences. Full results of these analyses are also now included as a supplement.

Page 7 Paragraph 2 Line 8: One or two-tailed test for p value is to be stated.

The use of two-tailed tests is now specified.

According to Cohen (pages 285-286 of Ref. 32), f = 0.10 (not 0.15) is considered a “small” effect size. The sentence in question has therefore been revised to read “According to Cohen [32], this would be between a “small” (f = 0.10) and a “medium” (f = 0.25) effect size.”

Page 7, Paragraph 2: Missing data (if any) is to be reported. If there is no missing data, a statement indicating this is to be included.

This has been addressed in the following paragraph.

Page 8 Paragraph 2 Line 5: Indicate clearly the p value in Figure 2D.

The P value is clearly shown at the top right of panel D, as well as stated in the text.

Please see below.

In response to the reviewer’s comments, P values for isometric/isokinetic torque have been moved from the text to the table, and interaction as well as main effects provided. These changes have made the footnote redundant, so it has been deleted. Conversely, main and interactions effects for Vmax and Pmax have been added to the text for completeness.

Attachment

Submitted filename: Response_to_Reviewers_auresp_2.docx

pone.0312961.s005.docx^{(16.9KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0312961.r006

Decision Letter 2

Ajaya Bhattarai

8 Dec 2024

PONE-D-24-32002R2Pilot randomized trial of the effect of antibacterial mouthwash on muscle contractile function in healthy young adultsPLOS ONE

Dear Dr. Coggan,

Please submit your revised manuscript by Jan 22 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.

Please include the following items when submitting your revised manuscript:

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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'.

We look forward to receiving your revised manuscript.

Kind regards,

Ajaya Bhattarai

Academic Editor

PLOS ONE

Journal Requirements:

Additional Editor Comments:

Minor revision is needed.

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #4: (No Response)

**********

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

Reviewer #3: Partly

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #3: Minor comment

For Table 1, based on CONSORT statement, statistical tests to compare baseline characteristics between intervention groups are not recommended. Instead, the CONSORT guidelines recommend presenting the baseline data descriptively. As such statistical tests/p values are to be omitted.

Reviewer #4: The authors in the study examined the effect of antibacterial mouthwash on muscle contractile function in healthy young adults. This study is well done and the manuscript has been revised based on reviewer comments.

I have only two minor comments

P.6. An examination of saliva samples. Typically, during ELISA analysis, duplicate sample analysis is employed for data evaluation. It is essential to incorporate these data regarding the coefficient of variations.

Results

You should add about F value in results of analysis of ANOVA.

**********

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.

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Reviewer #3: No

Reviewer #4: No

**********

PLoS One. 2025 Feb 12;20(2):e0312961. doi: 10.1371/journal.pone.0312961.r007

Author response to Decision Letter 3

7 Jan 2025

Reviewer #3: Minor comment

Statistical comparisons between groups have been removed.

I have only two minor comments

The CV for these triplicate determinations has now been stated.

Results

You should add about F value in results of analysis of ANOVA.

Added.

Attachment

Submitted filename: Response_to_Reviewers_auresp_3.docx

pone.0312961.s006.docx^{(13.4KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0312961.r008

Decision Letter 3

Ajaya Bhattarai

14 Jan 2025

PONE-D-24-32002R3Pilot randomized trial of the effect of antibacterial mouthwash on muscle contractile function in healthy young adultsPLOS ONE

Dear Dr. Coggan,

Please submit your revised manuscript by Feb 28 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.

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'.

We look forward to receiving your revised manuscript.

Kind regards,

Ajaya Bhattarai

Academic Editor

PLOS ONE

Journal Requirements:

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

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

Reviewer #4: All comments have been addressed

**********

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

Reviewer #3: Partly

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

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

Reviewer #3: Yes

Reviewer #4: Yes

**********

6. Review Comments to the Author

Reviewer #3: Minor comment

Table 1 footnote: The statement 'P values calculated using Fisher’s exact test and two-tailed unpaired t tests, as

appropriate.' is to be omitted.

Reviewer #4: (No Response)

**********

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.

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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 #3: No

Reviewer #4: No

**********

PLoS One. 2025 Feb 12;20(2):e0312961. doi: 10.1371/journal.pone.0312961.r009

Author response to Decision Letter 4

14 Jan 2025

Reviewer #3: Minor comment

Table 1 footnote: The statement 'P values calculated using Fisher’s exact test and two-tailed unpaired t tests, as appropriate.' is to be omitted.

The leftover footnote has been removed. (Why the reviewer insisted on further holding acceptance/publication of the paper versus simply letting this be handled at the copyediting stage is completely beyond me.)

Attachment

Submitted filename: Response_to_Reviewers_auresp_4.docx

pone.0312961.s007.docx^{(13.1KB, docx)}

PLoS One. doi: 10.1371/journal.pone.0312961.r010

Decision Letter 4

Ajaya Bhattarai

16 Jan 2025

Pilot randomized trial of the effect of antibacterial mouthwash on muscle contractile function in healthy young adults

PONE-D-24-32002R4

Dear Dr. Coggan,

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.

An invoice will be generated when your article is formally accepted. Please note, if your institution has a publishing partnership with PLOS and your article meets the relevant criteria, all or part of your publication costs will be covered. Please make sure your user information is up-to-date by logging into Editorial Manager at Editorial Manager® and clicking the ‘Update My Information' link at the top of the page. If you have any questions relating to publication charges, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Ajaya Bhattarai

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #3: All comments have been addressed

**********

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

Reviewer #3: Partly

**********

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

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

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

Reviewer #3: Yes

**********

6. Review Comments to the Author

Reviewer #3: (No Response)

**********

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 #3: No

**********

PLoS One. doi: 10.1371/journal.pone.0312961.r011

Acceptance letter

Ajaya Bhattarai

PONE-D-24-32002R4

PLOS ONE

Dear Dr. Coggan,

I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now being handed over to our production team.

At this stage, our production department will prepare your paper for publication. This includes ensuring the following:

* All references, tables, and figures are properly cited

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on behalf of

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PLOS ONE

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Data. Statistical analyses.

(DOCX)

pone.0312961.s001.docx^{(154KB, docx)}

S2 Data. Mouthwash study data (final).

(XLSX)

pone.0312961.s002.xlsx^{(76.3KB, xlsx)}

Attachment

Submitted filename: Response to Reviewers.docx

pone.0312961.s004.docx^{(21.7KB, docx)}

Attachment

Submitted filename: Response_to_Reviewers_auresp_2.docx

pone.0312961.s005.docx^{(16.9KB, docx)}

Attachment

Submitted filename: Response_to_Reviewers_auresp_3.docx

pone.0312961.s006.docx^{(13.4KB, docx)}

Attachment

Submitted filename: Response_to_Reviewers_auresp_4.docx

pone.0312961.s007.docx^{(13.1KB, docx)}

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.

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PERMALINK

Pilot randomized trial of the effect of antibacterial mouthwash on muscle contractile function in healthy young adults

Edgar J Gallardo

William S Zoughaib

Ahaan Singhal

Richard L Hoffman

Andrew R Coggan

Roles

Abstract

Introduction

Methods

Participants

Fig 1. CONSORT diagram.

Experimental design and protocol

Table 1. Participant characteristics.

Statistical analyses

Results and discussion

Markers of NO bioavailability

Fig 2. Effect of NaCl- or cetylpyridinium-containing mouthwash on markers of NO bioavailability.

Muscle contractile function

Table 2. Effect of NaCl- or cetylpyridinium-containing mouthwash on the maximal isometric/isokinetic torque of the knee extensors.

Fig 3. Effect of NaCl- or cetylpyridinium-containing mouthwash on maximal knee extensor velocity (Vmax) and power (Pmax).

Conclusions

Supporting information

Data Availability

Funding Statement

References

Author response to Decision Letter 0

Decision Letter 0

Ajaya Bhattarai

Roles

Author response to Decision Letter 1

Decision Letter 1

Ajaya Bhattarai

Roles

Author response to Decision Letter 2

Decision Letter 2

Ajaya Bhattarai

Roles

Author response to Decision Letter 3

Decision Letter 3

Ajaya Bhattarai

Roles

Author response to Decision Letter 4

Decision Letter 4

Ajaya Bhattarai

Roles

Acceptance letter

Ajaya Bhattarai

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

PERMALINK

RESOURCES

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