Can molecular hydrogen supplementation enhance physical performance in healthy adults? A systematic review and meta-analysis

Background Physical exertion during exercise often leads to increased oxidative stress and inflammatory responses, significantly affecting physical performance. Current strategies to mitigate these effects are limited by their effectiveness and potential side effects. Molecular hydrogen (H₂) has gained attention for its antioxidant and anti-inflammatory properties. Studies have suggested that H2 supplementation contributes to antioxidant potential and anti-fatigue during exercise, but the variance in the observations and study protocols is presented across those studies. Objective This systematic review and meta-analysis aimed to comprehensively characterize the effects of H₂ supplementation on physical performance (i.e., endurance, muscular strength, and explosive power), providing knowledge that can inform strategies using H2 for enhancing physical performance. Methods We conducted a literature search of six databases (PubMed, Web of Science, Medline, Sport-Discus, Embase, and PsycINFO) according to the PRISMA guidelines. The data were extracted from the included studies and converted into the standardized mean difference (SMD). After that, we performed random-effects meta-analyses and used the I2 statistic to evaluate heterogeneity. The Grading of Recommendations Assessment, Development, and Evaluation (GRADE) was used to assess the quality of the evidence obtained from this meta-analysis. Results In total, 27 publications consisting of 597 participants were included. The search finally included aerobic endurance, anaerobic endurance, muscular strength, lower limb explosive power, rating of perceived exertion (RPE), blood lactate (BLA), and average heart rate (HRavg) in the effect size (ES) synthesis. The ES of H2 on aerobic endurance, including V̇O2max (SMD = 0.09, p = 0.394; I2 = 0%) and aerobic endurance exercise (SMD = 0.04, p = 0.687; I2 = 0%), were not significant and trivial; the ES of H2 on 30 s maximal anaerobic endurance (SMD = 0.19, p = 0.239; I2 = 0%) was not significant and trivial; the ES of H2 on muscular strength (SMD = 0.19, p = 0.265; I2 = 0%) was not significant and trivial; but the ES of H2 on lower limb explosive power (SMD = 0.30, p = 0.018; I2 = 0%) was significant and small. In addition, H2 reduces RPE (SMD = −0.37, p = 0.009; I2 = 58.0%) and BLA (SMD = −0.37, p = 0.001; I2 = 22.0%) during exercise, but not HRavg (SMD = −0.27, p = 0.094; I2 = 0%). Conclusion These findings suggest that H2 supplementation is favorable in healthy adults to improve lower limb explosive power, alleviate fatigue, and boost BLA clearance, but may not be effectively improving aerobic and anaerobic endurance and muscular strength. Future studies with more rigorous designs are thus needed to examine and confirm the effects of H2 on these important functionalities in humans. Systematic review registration http://www.crd.york.ac.uk/PROSPERO.

Physical performance, including endurance, muscle strength, and explosive power, is the cornerstone of achievement in sports for non-athletic populations or athletes (1,2).It not only contributes to improving athletes' competitive performance on the field but also provides motivation for healthy adults to participate in sports (3)(4)(5).Oxidative stress occurs when the oxygen metabolism is produced and accumulated, eventually going beyond oxidation-resist ability (6,7).Studies have shown that physical activity of various intensities alters the levels of various oxidative biomarkers (8,9).However, physical exercise, especially with moderate-to high-intensity exertion, could lead to excessive oxidative stress, which may negatively impact redox homeostasis, worsen fatigue, and ultimately reduce physical performance (10)(11)(12)(13).Therefore, efforts have been put into exploring potential antioxidant approaches, which can thus help develop appropriate strategies to enhance physical performance (13)(14)(15).
Molecular hydrogen (H 2 ) is a promising antioxidant that selectively reduces hydroxyl radicals (•OH) and peroxynitrite (ONOO-) in cells without leading to a reduction of other reactive substances such as superoxide (O 2 -), hydrogen peroxide (H 2 O 2 ), and nitric oxide (NO) (16)(17)(18).Studies have shown that H 2 molecular, which can be delivered via different forms (i.e., H 2 gas and water, and intravenous H 2 -saline), can penetrate cell membranes and diffuse rapidly into organelles (e.g., mitochondria) (19), thus enhancing mitochondria functional performance (e.g., respiration and enzyme activity) and promoting ATP production or lactate oxidation (20,21).More recently, human studies have emerged to explore the potential benefits of using H 2 for physical performance in healthy adults and showed great promise of the H 2 -based intervention to improve physical performance (22)(23)(24)(25).However, the observations and protocol design across these studies on the effects of H 2 on physical performance were inconsistent.For example, some studies have observed that H 2 -rich water (HRW) supplementation before exercise could effectively increase maximal oxygen uptake (VȮ 2max ), anaerobic endurance, muscle strength, and lower limb explosive power in healthy adults (26)(27)(28), but other studies have shown contradictory findings (29)(30)(31).These inconsistencies may arise from the variance in participant characteristics, the protocol of H 2 administration, and types of exercise across studies.Only one previous review by Kawamura et al. (32) summarized the observations from only six studies and suggested that the validity of the observations from that literature should be examined and confirmed due to the very small number of included studies.Since then, many new studies have been performed to examine the effects of H 2 on endurance, muscle strength, and explosive power (24-26, 33, 34).Therefore, it is urgently demanded to more comprehensively characterize and explicitly examine the effects of H 2 on physical performance in healthy adults by summarizing the results of the most up-to-date publications.
We have thus conducted a systematic review and meta-analysis based on the available peer-reviewed publications.Only studies with randomized controlled or crossover designs are included, and several subgroup analyses are performed with the goal of providing critical knowledge of the appropriate design of H 2 -based intervention design for the improvement of physical performance.

Methods
This systematic review and meta-analysis were performed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analysis guideline (35) and registered with PROSPERO (ID CRD42022351559).

Data sources and search strategies
Two authors (K.Z. and Z.S.) independently searched PubMed, Web of Science, Medline, Sport-Discus, Embase, and PsycINFO databases from inception to 10 May 2024.The keywords of the search were as follows: "molecular hydrogen, " "hydrogen rich water, " "hydrogen-rich water, " "hydrogen rich saline, " "hydrogen-rich saline, " "hydrogen gas, " "hydrogen inhalation, " "hydrogen bathing, " "hydrogen-rich calcium powder, " "physical performance, " "athletic performance, " "exercise performance, " "physical exercise, " "aerobic performance, " "aerobic capacity, " "anaerobic performance, " "intermittent exercise, " "sprint, " "strength training, " and "resistance training" (The detailed search strategy is shown in Supplementary Table S1).In addition, a manual search was performed based on the reference lists of selected articles.The search was limited to English only, and no date restrictions were applied.

Selection criteria
To be included in this systematic review, previous studies must meet the following eligibility criteria in accordance with PICOS.
1. Participants: the participants were healthy adults with a mean age of ≥18 years and were free from any dietary supplements or medications while the experiment lasted; 2. Intervention: the intervention was the supplementation of H 2 by the participants.The source of H 2 was not limited; 3. Comparator/Control: the control group used placebos that were identical in appearance, texture, and flavor to H 2 products (e.g., drinking water, air, and capsules); 4. Outcomes: the outcomes include at least one of the measures related to physical performance (e.g., aerobic and anaerobic endurance, muscular strength, lower limb explosive power, subjective fatigue, blood lactate (BLA), and heart rate); 5. Study design: the design of the study was a randomized crossover or randomized controlled trial.
Articles were excluded if they fulfilled the following criteria: 1) animal trials; 2) written in a language other than English or unable to obtain outcome data; 3) review papers and conference articles; and 4) repeated publications.

Data extraction and outcomes
According to the Cochrane Collaboration Handbook, the data extraction process was conducted independently by two authors (C.Y. and Z.S.) (36).The extracted information from the publications included the following: the study (authors and year), sample size, participants (age, height, weight, sex, and training status), methods of H 2 administration, exercise protocol, and outcome measures.Any outcome measures on which the two authors disagreed were discussed with the other two authors (J.Z. and D.B.) until a consensus was achieved.
The mean and standard deviation of each outcome in post-tests were extracted for each included study.If the post-test values were not available, they were calculated using the following formulas, where the correlation coefficient (Corr) was set at 0.5 (36,37).
If relevant data were missing, we emailed the corresponding author or other authors to request it (36).We extracted relevant data using WebPlotDigitizer (version 4.6) for studies when the data could not be obtained by contacting the authors (38).
Based on the included studies, aerobic endurance, anaerobic endurance, muscular strength, and lower limb explosive power performance were ultimately incorporated into the data synthesis.
The primary outcome of aerobic endurance performance was maximum oxygen uptake (VȮ 2max ) during an incremental load exercise test or peak oxygen uptake (VȮ 2peak ) when VȮ 2max was not available (39,40).The secondary outcome of aerobic endurance performance was aerobic endurance exercise performance, for example, time-to-exhaustion (TTE) or power during incremental load exercise test or fixed-load submaximal test; the time or speed in time trial test (TT).
The primary outcome of anaerobic endurance performance was power output during the 30 s maximal anaerobic test.
The primary outcome of muscle strength was peak torque or force in the maximal voluntary isometric strength test (MVIS) or maximal isokinetic strength test performed pre-or post-highintensity exercise.
The primary outcome of lower limb explosive power was countermovement jump (CMJ) height, time of short sprint, or peak power output during 10 s maximal effort exercises.
The exploratory outcomes were the rating of perceived exertion (RPE), BLA, and average heart rate (HR avg ) during physical performance.The RPE, BLA, and HR avg are widely used and are important metrics to characterize subjective fatigue, intensity, and physiologic stress that are closely associated with physical performance (41)(42)(43).By exploring the effects of H 2 on them, it will help more comprehensively characterize the effects of H 2 supplementation on physical performance.

Quality assessment
Two authors independently evaluated the risk of bias in included studies using the Cochrane Collaboration's tool (44), which contains six items: 1) selection bias; 2) performance bias; 3) detection bias; 4) attrition bias; 5) reporting bias; and 6) other bias.Each item is categorized into three levels: low-risk bias (green), unclear risk bias (yellow), and high-risk bias (red).Studies were defined as having a high-risk bias if ≥1 item had a high-risk bias.The risk of bias is low if all items are assessed as low risk of bias.Others were assessed as moderate risk of bias.Additionally, the quality of evidence for outcomes was evaluated using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) (45,46).The quality of the GRADE evidence was graded as high, moderate, low, and very low based on the quality of study design, quality of implementation, uncertainty of results, and consistency of results (45).
The Funnel plots and Egger tests were used to evaluate publication bias.If potential publication bias was detected, we used the trim and fill method for the sensitivity analysis of the results (50).All the statistical significance was set at a p-value of <0.05.

Study selection
The screening procedure of the included studies is shown in Figure 1.A total of 401 potentially relevant publications were retrieved (PubMed n = 77, SPORT-Discus n = 65, Medline n = 71, Web of Science n = 89, PsycINFO n = 5, and Embase = 94).Based on the criteria above, 248 publications were discharged after reviewing the titles and abstracts.After evaluating the full texts, 27 publications (29 studies) were included in the systematic review.Finally, 25 publications consisting of 27 studies (23 randomized crossover designs and 4 randomized controlled trials) were included in the quantitative synthesis (Table 1).One study (28) included two randomized controlled trials, and the other study (23) included a randomized crossover trial and a randomized controlled trial.

Participant characteristics
A total of 597 participants, with mean ages ranging from 17.5 to 51.5 years, were included.The training status of these participants was classified according to the included studies as untrained (n = 224) and trained (n = 373), with 215 of them being well-trained athletes (e.g., professional soccer players and elite runners).

Methods of H 2 administration
A gold standard regimen for H 2 application does not appear to exist.The included studies implemented four sources of H 2 , that is, drinking HRW (n = 18) ( 22

Exercise protocol and outcome measurements
The included studies highlighted the effects of H 2 supplementation on aerobic endurance, anaerobic endurance, muscular strength, and lower extremity explosive strength in participants.In these studies, continuous incremental load and fixed-load subliminal exercise were the most commonly used aerobic endurance intervention or testing protocols.VȮ 2max , VȮ 2peak , TTE, race time, and power were metrics used to measure aerobic endurance performance (24,26,28,29,33,55,57,61,63).The 30 s maximal anaerobic power test (i.e., pedaling bicycle or rowing dynamometer) was used to assess the anaerobic endurance (i.e., mean or maximal power) (24,26,27).One study (57) used the MVIS to assess the force of knee extension prior to highintensity aerobic exercise; four studies (27,30,51,62) were conducted to evaluate the magnitude of knee extensor force or peak torque in the MVIC after vigorous exercise.Eight studies (25,27,30,34,61,(63)(64)(65) evaluated alterations in lower limb explosive power (i.e., CMJ height and peak power output during 10 s or 30 m sprint) during or after vigorous exercise in participants.One study (53) used the special fitness test to assess the effects of HRW intake on athletic performance in judo athletes.Additionally, the included studies focused on assessing the effects of H 2 administration on various physiological parameters during exercise, such as RPE, BLA, HR, pH, respiratory function, antioxidant levels, muscle oxygenation, and endocrine system.The outcomes of each study are summarized in Table 1.

Quality assessment
The risk of bias in the 27 publications (29 studies) was assessed, and a consensus was reached after discussion.The overall result is shown in Figure 2. Two studies (23, 28) did not adequately report on participant randomization and concealment methods.Five studies (23,24,30,55,60) did not adequately describe participant, staff, or evaluator blinding.No studies had incomplete results due to participants' withdrawal.All studies reported experimental procedures and conducted the experiments as planned.According to the possibility of bias, the study was assessed as being low risk, moderate risk, or high risk.One study (23) was evaluated as having a high-risk bias, five studies (24, 28, 30, 55, 60) had a moderate risk bias, and others were assessed as having a low-risk bias.The quality of evidence for outcomes was evaluated as moderate to high, and details for the evaluation of the GRADE framework are presented in Supplementary Table S2.

Meta-analysis
A subgroup analysis was performed on aerobic endurance, anaerobic endurance, muscle strength, lower limb explosive power, RPE, and BLA, considering potential sources of heterogeneity, including exercise types and H 2 sources.Additionally, we used a subgroup analysis to explore the effects of H 2 supplementation on muscle performance before or after vigorous exercise (Table 2).

Effects of H 2 on anaerobic endurance
One study (26) showed that H 2 can significantly improve mean and peak power output during the 30 s maximal anaerobic test as compared to the placebo.One study (24) showed that H 2 can significantly improve peak power output in a 30 s maximal anaerobic test compared to placebo but cannot significantly improve mean power, while another study (27) showed the opposite result that H 2 cannot significantly improve mean power output during the 30 s maximal anaerobic test (Table 1).S1H) and Egger's test (t = 1.26, p = 0.296) indicated that there was no publication bias.

Discussion
To our knowledge, this is the first systematic review and metaanalysis exploring the effects of H 2 supplementation on physical performance in healthy adults.The results suggest that H 2 supplementation is promising for improving lower limb explosive power and reducing RPE and BLA clearance during vigorous exercise.However, it does not enhance endurance performance and muscle strength or decrease HR avg .
This meta-analysis suggests that administering H 2 before or after exercise may serve as a potential strategy to effectively enhance lower limb explosive power in healthy adults.One potential mechanism underlying the effects of H 2 on explosive power is that H 2 can directly react with strong oxidants in vivo [e.g., hydroxyl radicals (•OH)] to modulate Ca 2+ or mitochondrial ATP-dependent K + channels, thus facilitating mitochondrial ATP production (20,(66)(67)(68)(69). Additionally, H 2 could reduce intracellular reactive oxygen species (ROS) levels and thus enhance muscle contractile function (27,70).For example, a study conducted on soccer players demonstrated that administering three successive doses of 500 mL of HRW prior to high-intensity aerobic exercise increased the mean power frequency of skeletal muscles during subsequent strength tests (51).However, this finding that H 2 promotes lower limb explosive power may be influenced by a small sample size (n = 92) or movement pattern.One example is that H 2 significantly improved participants' sprint performance compared to their vertical jump performance (25,63).Therefore, more research is still needed to confirm this finding in the future.The result showed that H 2 did not significantly improve muscle strength after aerobic endurance exercise.One possible reason is that intense aerobic exercise leads to a consumption of H 2 in the body that does not continually provide benefits for subsequent muscle strength performance.One study (34) shows that 1,260 mL of HRW intake can increase the movement velocity of multiple lunges during resistance training.Therefore, more studies are needed in the future to clarify the effects of H 2 supplementation on muscular strength performance in isolated resistance training.It has been observed that H 2 supplementation cannot significantly improve aerobic and anaerobic endurance performance.Endurance performance depends on the multiple factors of human respiratory function, oxygen transport, and local muscle oxygen utilization during exercise (71, 72).Studies have shown that using H 2 failed to significantly improve these critical factors (e.g., VȮ 2max and running economy) of endurance performance (23, 29,31,56,57), thus leading to the insignificant benefits of H 2 on this important function.
While H 2 supplementation does not appear to enhance endurance performance or increase muscle strength, it does demonstrate favorable effects in reducing RPE, BLA levels, and HR avg among individuals engaged in high-intensity exercise.H 2 appears to be a neuroprotective agent that facilitates the restoration of neuronal oxidative damage by reducing oxidative stress and neuroinflammation (16,(73)(74)(75).H 2 intake has also been reported to induce positive effects on exercise acidosis (56), thus modulating intracellular and extracellular buffering capacity during vigorous exercise (76).The decrease in BLA during exercise may be attributed to the fact that molecular H 2 accelerates the transport of BLA to the liver for storage and oxidation, as well as increasing the utilization of lactate as a fuel by the muscles (56,77).Subgroup analysis reveals that H 2 supplementation reduces BLA concentration in aerobic endurance exercise, which is superior to other exercise types.The reduction in BLA response during aerobic endurance exercise may indicate that H 2 supplementation enhances oxidative energy metabolism (28).Indeed, this finding may be unreliable due to the small number of studies on other exercise types.Therefore, future research should focus more on the effects of H 2 supplementation on anaerobic endurance, muscular strength, and repeated sprint performance.Subgroup analyses reveal two important factors that likely contribute to the effects of H 2 supplementation on RPE.First, we observed that the effects were greater in strength training and repeated sprints as compared to endurance exercise.The observed variations in RPE could be attributed to the disparities in the energy supply mechanisms across different types of exercises.It is plausible that H 2 gas may exhibit a higher affinity toward the phosphagen system when compared to the oxidative and glycolytic systems (66).Second, inhalation of H 2 gas (HRG) is superior to the ingestion of HRW in mitigating RPE.The observed discrepancy can be attributed to the fact that the respiratory absorption of molecular H 2 is significantly more efficient and comprehensive in comparison to its digestive absorption in HRW.Nonetheless, given the restricted sample size, it is imperative to ensure further validation of the outcomes of the subgroup analysis.

Limitations
Five included studies with a small number of participants (n ≤ 10) (23, 27, 30, 52, 53) may lead to potential bias.Most studies to date focus on only younger and middle-aged men, and future studies are highly demanded to examine the benefits of H 2 for women and those with older age.The current studies only investigated the effects of H 2 supplementation for 1-14 days and future studies need to focus on the effects of longer supplementation periods.Some studies did not report or detect H 2 concentrations, and the H 2 dosing regimen was highly variable.The dose-response relationship between H 2 and physical performance has not been established, which should be explored in the future to determine the most appropriate dosage and intervention protocol for H 2 for enhancing physical performance.

Conclusion
In summary, this systematic review and meta-analysis suggest that short-term (<14 days) H 2 supplementation protocols contribute to improved lower limb explosive power, fatigue relief, and BLA clearance but may not significantly improve aerobic endurance, anaerobic endurance, or muscular strength.Inhaling H 2 shows promise as the optimal method for improving physical performance (i.e., lower limb explosive power) in healthy adults.Future studies with rigorous designs are needed to help obtain more definitive conclusions on the effects of H 2 on lower limb explosive power and muscle strength in healthy adults.

FIGURE 2
FIGURE 2Risk of bias in the included studies.

FIGURE 6 Forest
FIGURE 6Forest plot of the effects of H 2 supplementation on muscle strength.

FIGURE 7 Forest
FIGURE 7Forest plot of the effects of H 2 supplementation on lower limb explosive power.

TABLE 1
Characteristics of the included studies (n = 29).

TABLE 2
Subgroup analysis results regarding the effects of H 2 on RPE and BLA.Forest plot of the effects of H 2 supplementation on VȮ 2max /VȮ 2peak .Exp.1, Experiment 1; Exp.2, Experiment 2.