Abstract

Introduction

Molecular hydrogen (H₂) was initially considered a biologically inactive gas. However, Dole et al. [1] published the first study (mouse model) reporting a significant regression of skin tumors in response to hyperbaric H₂ treatment. H₂ was shown to have strong selective antioxidative and anti-apoptotic properties, reducing oxidative stress through the scavenging of harmful cytotoxic hydroxyl radicals (OH●) [2]. Recently, Ichihara et al. [3] reported anti-apoptotic, anti-inflammatory, and antioxidative properties of H₂. H₂ was also considered to be a signaling molecule that contributed to modulation and regulation of gene expression [3]. An anti-fatigue effect of H₂ in response to pre-exercise intake of hydrogen rich water (HRW) was shown across different modes of exercise, specifically, repeated isokinetic knee extensions [4], intermittent cycling sprints [5], repeated running sprints [6], anaerobic performance [7], and strength-endurance drills [8]. Research also showed that an anti-fatigue effect of pre-exercise HRW intake seemed to be dependent on the current performance status of athletes [7, 9]. Further, studies demonstrated that pre-exercise H₂ exposure led to a lower rate of perceived exertion (RPE), enhanced ventilation efficiency, reduction in blood lactate concentration [10], and stimulated prefrontal cortex activity [11], particularly during higher exercise intensities. In addition, acute HRW intake resulted in a post-exercise lactate lowering effect [4, 12] and lower delayed onset of muscle soreness after strength exercise [8, 13]. However, pre-exercise HRW consumption did not positively affect submaximal running performance, physiological responses, or time to exhaustion, in response to a maximal incremental running test [14]. In addition, no significant difference was reported between HRW and control groups for time to exhaustion in an incremental cycling test in a heated environment [15], and there was no ergogenic effect of H₂ during an incremental maximal test in either amateur or professional cyclists [7]. Despite the inconsistent exercise ergogenic effects of H₂, recent in vitro studies have reported mitochondrial effects of H₂. H₂ was shown to increase mitochondrial oxygen consumption rate, stimulate mitochondrial Q cycle and enhance oxidative adenosine triphosphate production [16, 17].

Based on the potential aerobic energy system benefits of H₂, this study was designed to investigate whether acute HRW supplementation improved running performance at maximal aerobic speed. From a practical application standpoint, maximal aerobic speed is closely related to the running velocity that can be sustained by elite runners over 3000 m [18]. Therefore, the primary aim of this study was to assess the effect of acute pre-exercise HRW intake on time to exhaustion when running at maximal aerobic speed in trained track and field runners. We hypothesized that the anti-fatigue effect of pre-exercise HRW ingestion [4, 6, 8, 10] would improve running performance at maximal aerobic speed with an increase in time to exhaustion.

Methods

Results

Raw data are available in S1–S3 Tables. The anthropometric and performance characteristics of participants are presented in Table 1. Each participant received 567 μmol of H₂ dissolved in 1260 ml of HRW during the experimental running protocol. The dose relative to body mass was 8.26 ± 0.71 μmol⋅kg^-1 expressed as mean ± standard deviation or 8.17 (7.70, 8.80) μmol⋅kg^-1 expressed as median (first and third quartiles).

After logarithmic transformation, all variables listed in Table 3 had normal distributions (all p ≥ 0.073, Shapiro-Wilk test) except BF (p = 0.001). The departure from normality was considered small after visual inspection of the quantile-quantile plot and the BF variable was also processed using a t-test because it is considered robust for such departure from normality. The coefficient of variation for time to exhaustion estimated from this crossover study was 13% with a 95% confidence interval of 10 to 18%. The effects of HRW on performance and physiological variables are presented in Table 3. No statistically significant effects of HRW were found (all p ≥ 0.20, paired t-test). Absolute Cohen’s d values ranged from 0.01 to 0.19, indicating trivial effects. Analysis of individual responses of time to exhaustion (Fig 3) revealed that 12 runners responded positively to HRW, 3 runners did not respond, and 9 runners responded negatively. The odds ratio of positive/negative responders (12/9) was not significant (p = 0.51, chi-square test). Therefore, the hypothesis that acute HRW supplementation has an ergogenic effect in trained runners running at maximal aerobic speed was rejected.

Two candidate moderators, namely age and maximal aerobic speed (Table 1), had non-normal distributions, so correlation analysis was performed using the non-parametric Spearman’s correlation coefficient. Values of Spearman’s correlation coefficient ranged from −0.28 to 0.30 and these values were not statistically different from zero (all p ≥ 0.16, Table 4). Specifically, runner performance level expressed as pooled time to exhaustion (average of HRW and placebo values) could not be considered a suitable moderator of the effect of HRW on time to exhaustion (r = 0.12, p = 0.56, Fig 4). Thus, no suitable moderator of the effect of HRW was found in this study.

Discussion

The primary aim of this study was to assess the influence of a dose of 1260 ml HRW on running performance as indicated by time to exhaustion at maximal aerobic speed in trained track and field runners. Based on previous studies that have demonstrated an anti-fatigue effect of HRW intake before exercise [4–6, 8, 10, 12], we hypothesized that HRW intake prior to exercise would improve maximal aerobic speed performance, specifically running time to exhaustion. Contrary to our hypothesis, we observed no significant effect of HRW in time to exhaustion, as a valuable predictor of 1500 m running performance [30], or any other physiological variable during running at maximal aerobic speed.

It is accepted that running to exhaustion at maximal aerobic speed represents a very high exercise intensity, where a substantial anaerobic glycolytic contribution towards adenosine triphosphate resynthesis is involved, and consequently, there is an onset of blood lactate accumulation [31]. In the current study, trained track and field runners ran at a determined speed of 18.3 ± 1.5 km⋅h^-1 for a duration of 217 ± 49 s after ingesting HRW, and for 227 ± 53 s, after ingesting a placebo. This running velocity corresponds with a time to exhaustion ranging from 2.5 to 10 min for different kinds of activities [32], while it is slower compared with elite and sub-elite middle- and long-distance runners [32, 33]. From a practical standpoint, if maximal aerobic speed and time to exhaustion describes 95% of variance in the average race velocity over 1500 m [30], we suggest that the enhancement for middle-distance performance by acute supplementation of 1260 ml HRW would also be ineffective. In addition, the post-exercise blood lactate concentration was 9.9 ± 2.2 mmol⋅L^-1 and 10.1 ± 2.0 mmol⋅L^-1 for HRW and placebo, respectively. These values indirectly indicated exercise-induced metabolic acidosis [34] and potential peripheral fatigue development [35]. Besides the increasing muscle acidosis, an excessive production of reactive oxygen species during exhaustive running [36, 37] may have also contributed to the deterioration of muscle performance, as previously demonstrated [38].

The results show that the expected antifatigue effect of H₂ was not detected when a dose of 1260 ml HRW (H₂ = 0.9 ppm) was ingested prior to the run at maximal aerobic speed with a duration of up to ~230 s. We assume that the anticipated antifatigue effects of H₂ was likely prevented by the increasing muscle acidosis, together with excessive oxidative stress. This result is consistent with some previous studies that examined the acute ingestion of HRW before exercise. For instance, an acute intake dose of 290 ml HRW (H₂ = 1.0 ppm) before testing and 290 ml HRW during a 10 min rest period between submaximal and maximal sections of an experimental protocol did not affect cardiorespiratory and metabolic variables during an incremental submaximal running test (34–91% VO₂max) or the subsequent ~619 s running to exhaustion during incremental exercise [14]. These authors concluded that two doses of 290 ml of HRW before incremental running to exhaustion was not sufficiently ergogenic in endurance-trained athletes [14]. Similarly to our findings, Ito et al. [15] reported that an applied dose of 2.0 ml⋅kg^-1 of either HRW or placebo, ingested every 15 min within 60 min of cycling at 65% of VO₂max, followed by an incremental cycling test to exhaustion, did not improve performance in well trained triathletes. In this context, Nogueira et al. [39] reported no significant changes in running time to exhaustion in rats after inhaling either 2% H₂ or H₂ free air during acute, exhaustive physical exercise. Interestingly, despite no ergogenic effect of H₂ inhalation on running performance, post-exercise biochemical analysis revealed important findings, where H₂ inhalation was associated with effective downregulation of muscle damage, reducing oxidative stress, inflammation, and apoptosis after exhaustive acute exercise in the rats that were unaccustomed to this level of exercise [39].

From methodological standpoint, the question arises whether our results may be tainted by small sample size or poor reliability of measurement. Before starting the experimental part, we performed a power analysis based on the results of Botek et al. [10]. From this analysis the desired sample size was calculated to be 24 participants, which was honored in the experiment. Previous studies [4, 5, 10] used even smaller sample sizes [8, 10 and 12, respectively] and found that HRW improved responses to exercise, including a reduction in lactate concentration and fatigue. Therefore, the sample size in this study is unlikely to be too small. The reliability of the time to exhaustion in this study was 13%, expressed as a coefficient of variation. Although this value was obtained from an intervention study and not a reliability study, it is comparable to values in reliability studies: 11% [32], 13% [40], indicating a good level of methodology and standardization of measurement in our laboratory. The coefficient of variation of time to exhaustion in constant-power tests is known to be highest in physical performance tests [28]. However, Hopkins et al. [41], using the relationship between exercise duration and power output, showed that the reliability of the equivalent mean power calculated from the constant-power test (0.6%) is even better than from the constant-work test (1.0%). We therefore consider time to exhaustion as a suitable index for tracking changes in performance.

Contrary to our current findings, there are four studies that have reported an anti-fatigue or performance enhancing effect of HRW intake prior to exercise. Specifically, an antifatigue effect of HRW (2 L per day for 2 weeks pre-exercise, dissolved H₂ = 0.15 to 0.45 ppm, and pH = 9.8.) during intermittent cycling was demonstrated by Da Ponte et al. [5], who reported a 7.4% attenuation in the decline of peak power output from the 6^th to the 9^th of 10 sprints. In addition, in soccer players, Aoki et al. [4] reported an attenuated decrease (3.7%) in peak torque for 20 isokinetic knee extensions, following 30 min of cycle ergometry at an intensity of 75% VO₂, when 1.5 L of HRW (H₂ = 1.84–2.04 ppm) was ingested within 8 hours pre-exercise. Positive effects of acute, intermittent ingestion of HRW on resistance training was recently demonstrated by Botek et al. [8]. Ingestion of 1260 ml HRW (H₂ = 0.9 ppm) pre-exercise resulted in lower blood lactate concentration, improve muscle function, and alleviated muscle pain perception in physical active males.

Importantly, it is very difficult to accurately compare our findings with the previously mentioned studies due to the variable methodology used, including exercise protocols (mode, intensity, duration), dose of HRW and its chemical properties (concentration of dissolved H₂, and its pH), and the training status of involved participants. These variables all impact the body response to a particular mode of exercise. For instance, Botek et al. [9] recently showed that pre-race hydration with 1680 ml of HRW improved endurance running performance by 1.3% in the slowest runners, whilst the effect of HRW on race performance in the fastest runners was unclear (deterioration by 0.8%), and concluded that the magnitude of anti-fatigue effect of H₂ depended on individual adaptation level. The results suggested that faster athletes seem to be less sensitive to acute H₂ supplementation compared with slower athletes who exhibit higher benefits from acute H₂ intake. In contrast, Timón et al. [7] recently reported an improved anaerobic performance in a group of trained cyclists after one week of HRW administration (H₂ = 1.9 ppm, dose of ~2 L per day) compared with no improvements in a group of amateur cyclists. In the current study, we did not find a suitable moderator of individual responses of time to exhaustion, which may have been due to the homogeneity of running performance in our cohort of runners. A future study involving athletes with more heterogenous performance is needed to assess this phenomenon.

Considering our results and previous research, we suggest that acute H₂ intake before exercise does not always provide ergogenic effects. Based on current results, we could not recommend acute pre-exercise HRW intake as an ergogenic supplement to improve time to exhaustion at maximal aerobic speed. There are some limitations and issues regarding HRW application in this study. 1) oxidative stress variables were not assessed. It appears that this information may be helpful for a deeper understanding of how H₂ may affect ROS production and performance responses. 2) the dosage of H₂ was constant for all participants for logistical reasons and was not adjusted to body mass. 3) Several variables were compared, and no technique was used to control for Type 1 statistical error.

Conclusions

This study found that a dose of 1260 ml HRW ingested prior to an exhaustive run had no significant effect on the running performance at maximal aerobic speed in a cohort of the national level track and field runners. Acute ingestion of HRW as a hydration strategy prior to exercise in trained middle-distance runners is not recommended to improve performance.

Supporting information

Funding Statement

This study was supported by the Palacký University Olomouc (URL: www.upol.cz), grant project IGA_FTK_2020_011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Data Availability

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

References