Hydrogen-rich water enhances exercise performanceScientific Research

INTRODUCTION

Many previous studies have shown the beneficial effects of the intake of hydrogen (H₂)-rich water (HW).¹² For instance, the intake of HW was shown to stimulate lipid metabolism by inducing fibroblast growth factor 21 expression and/or peroxisome proliferator-activated receptor-γ coactivator-1α expression in diabetic db/db mice.³⁴ Furthermore, HW consumption improved lipid and glucose metabolism in patients with type 2 diabetes,⁵ and reduced the body fat percentage and serum triglyceride levels in middle-aged overweight women.⁶

Recently, the effectiveness of HW has been demonstrated by studies regarding exercise physiology and sports and health sciences.⁷⁸ For example, Sha et al.⁹ revealed that a 2-month intake of HW enhanced the antioxidant activity in female soccer players. Ara et al.¹⁰ demonstrated that the increased antioxidative activities induced by loading of HW attenuated exercise-induced chronic fatigue in mice. Aoki et al.¹¹ indicated that the intake of HW suppresses acute fatigue as well as an increase in blood lactate (La) levels during exercise in elite athletes. Additionally, the findings that HW administration relieved exercise-induced psychometric fatigue¹² and maintained the peak power output in repetitive sprints¹³ have also been reported in humans.

The supplementation of HW enhances mitochondrial ATP production, suggesting that intake of HW increase aerobic metabolism.¹⁴ Therefore, it can be expected that HW supplementation would increase the aerobic capacity. In fact, LeBaron et al.¹⁵ speculated that the intake of HW improved oxygen extraction and utilisation in active skeletal muscles based on the decreased heart rate (HR) observed during the same exercise intensity as a placebo trial in humans. However, the effects of HW on aerobic capacity during exercise are not clear. The purpose of the present study, therefore, was to clarify the effects of a single or a 2-week continuous supplementation of HW on aerobic capacity during an incremental cycling exercise in humans.

PARTICIPANTS AND METHODS

Experimental design

This study completed at our laboratory in Chubu University consisted of two experiments with a single-blind method design (Figure 1). In the first experiment, participants ingested, at random, HW or placebo water (PW) before performing an exercise test to investigate the effects of single supplementation of HW on aerobic capacity (experiment [Exp] 1). In the second experiment, we randomly divided the participants into the following two groups: HW and PW groups. The participants performed the same exercise test as Exp 1 before and after 2 weeks of HW intake to elucidate the chronic effect of HW on aerobic capacity (Exp 2).

Participants and ethical approval

We orally recruited healthy participants at Chubu University, who were able to perform an incremental cycling exercise test. Six male and three female university students volunteered to participate in Exp 1 (Table 1). Twenty male university students participated in Exp 2, and were divided into two experimental groups: HW (n = 10) and PW groups (n = 10) (Table 1).

Each participant was informed of the experimental protocol and the possible risks involved in this study before providing written consent. This study protocol was approved by the Ethical Committee of Chubu University (approved No. 260086-2) on March 29, 2018.

Experimental protocol

In Exp 1, the participants came to our laboratory and drank 500 mL of HW or PW. After 30 minutes of seated rest, they performed an incremental cycling exercise test. They underwent the exercise test twice at random, i.e., HW and PW trials, with at least 24 hours between trials for recovery.

In Exp 2, the participants performed the exercise test twice, once before and once again at 2 weeks after the intake of HW or PW. The participants drank 500 mL of HW or PW on all weekdays, i.e., they drank HW or PW 10 times with a total volume of 5 L. The post exercise test was performed at 30 minutes after drinking the experimental water, as in Exp 1.

Preparation of HW

A stick-shaped H₂ generator (Hydrogen Water 7.0; MiZ Co. Ltd., Kanagawa, Japan) and 500-mL plastic bottles were used to prepare HW. HW was administrated in the laboratory at room temperature for 24 hours after the H₂ generator was immersed in the bottle containing water. HW was stirred just before drinking to dissolve the H₂, and the same procedure was done for PW to preserve the single-blind experimental design.

The concentration of dissolved H₂ in water was measured by titration with a dissolved H₂ reagent methylene blue kit (MiZ Co. Ltd., Kanagawa, Japan), as described previously.¹⁶ The measured concentration was 4.3 ± 0.9 ppm in Exp 1 and 5.9 ± 0.2 ppm in Exp 2.

Measurement of peak oxygen uptake and peak load

All participants underwent a practice session in advance to become accustomed to the exercise test. We adopted an incremental cycling exercise test using a bicycle ergometer (Aerobike 75XLIII; Combi Wellness Corporation, Tokyo, Japan). The workload was gradually increased by 20 W every 1 minute. As shown in Figure 2, the participants performed the exercise after a 3-minute warm up at 0 W following a 5-minute rest on the ergometer with a respiratory mask. The participants kept the pedalling cadence of 60 r/min during the exercise and performed the exercise until they could not maintain a pedalling rate of 50 r/min. We informed the participants that they were unable to return the cadence to 60 r/min regardless of the experimenters’ verbal exhortation.

We measured the oxygen uptake (VO₂), carbon dioxide output (VCO₂), minute ventilation (V_E), and HR on a breath-by-breath basis using a metabolic gas analyser (AE-310S; Minato Medical Science, Osaka, Japan). The respiratory exchange ratio (RER) was calculated from the ratio of VO₂ and VCO₂. VO₂ was averaged every 20 seconds and peak VO₂ (VO_2peak) was defined as the peak value of the averaged VO₂ during the exercise. The peak values of other respiratory and circulatory parameters were calculated in the same way. We also recorded the rate of perceived exertion (RPE) using the Borg Scale¹⁷ for every minute during the exercise. The workload at the end of the exercise was determined as the peak load. In the present study, VO_2peak divided by body mass and peak load were used as parameters of aerobic capacity.

Evaluation of blood La, oxidative stress, and antioxidant activity

We obtained a blood sample from the participants’ fingertips before, during (at 150 W), and immediately after the exercise to evaluate the La concentration, oxidative stress, and antioxidant activity. The La concentration was measured using a portable lactate measuring device (Lactate pro2; Arkray, Kyoto, Japan). To assess the oxidative stress and antioxidant activity, plasma was obtained by centrifugation, and the Free Radical Elective Evaluator (FREE^® Carpe Diem; Wismerll, Tokyo, Japan) was used to assess the diacron reactive oxygen metabolites (d-ROMs), which optically measures the blood concentration of hydroperoxides according to the optical measurement method.¹⁸ The biological antioxidant potential (BAP) was also assessed, which evaluates the antioxidant activity by measuring the capacity to reduce Fe³⁺ to Fe²⁺.¹⁹²⁰ The values of d-ROMs are expressed in UCARR, which is an arbitrary unit (1 UCARR corresponds to 0.08 mg/dL H₂ O₂).²¹ The details of the mechanisms and procedures of d-ROMs and BAP tests have been previously described.²²

Statistical analysis

Sample size calculation was performed by using the G* Power 3.1.9.7 software (Heinrich-Heine-Universität, Düsseldorf, Germany). The primary outcome variable in this study was the change in VO_2peak, peak load, and oxidative stress by drinking HW. A minimal sample size of eight participants in Exp 1 and ten participants in each group in Exp 2 was respectively needed for a statistical power of 80% (1 – β), effect size of 0.35, and an α error rate of 0.05 in the case of using a two-way repeated measures analysis of variance (ANOVA).

In Exp 1, a paired t-test was used to compare peak loads, respiratory and circulatory parameters, and RPE between the HW and PW trials. A two-way repeated measures ANOVA was used to assess the changes in La, d-ROMs, and BAP responses to the exercise. If a significant interaction was observed, an analysis of the simple main effect was conducted, and then, Bonferroni’s test for multiple comparisons was further used to identify the specific differences. When only the main effects were significant, the Bonferroni’s test for multiple comparisons was performed.

In Exp 2, a two-way repeated measures ANOVA was performed to compare peak load, respiratory and circulatory parameters, and RPE before and after 2 weeks of intake of experimental water between the HW and PW groups. When a significant interaction was observed, an analysis of the simple main effect was performed. Bonferroni’s test for multiple comparisons test was used when only the main effects were observed. Furthermore, an unpaired t-test was used to compare delta changes from before to 2 weeks after continuous intake of experimental water between the HW and PW groups. A two-way repeated measures ANOVA was also used to evaluate the changes in La, d-ROMs, and BAP during the exercise test, separately for each group. The post hoc analysis was the same as that of Exp 1.

Statistical analyses were carried out by using StatView 5.0 (SAS Institute, Cary, NC, USA) and SPSS 24.0 for Windows software (IBM, Armonk, NY, USA). The significance level was defined as P < 0.05. All values are presented as the mean ± standard error (SE).

RESULTS

Characteristics of participants in this study

The characteristics of participants in Exp 1 and 2 are shown in Table 1.

Effects of the single supplementation of HW (Exp 1)

Table 2 shows VO₂, VCO₂, RER, V_E, and HR at rest for each trial. There were no significant differences in these parameters between the two trials (P > 0.10). There were no significant differences in the peak values of VCO₂, RER, V_E, HR, and RPE Table 2 between the trials either (P > 0.10). As shown in Figure 3, HW did not significantly increase VO_2peak (P = 0.30) and peak load (P = 0.58). The exercise significantly increased La levels and BAP (P < 0.01) but not d-ROMs (P = 0.24); however, the significant effects of HW were not observed in each parameter (P > 0.10; Table 3).

Effects of 2-week continuous supplementation of HW (Exp 2)

An unpaired t-test showed no significant difference in height (P = 0.28), weight (P = 0.54), age (P = 0.87; Table 1), and baselines peak loads (P = 0.84) and VO_2peak (P = 0.43) before starting the intake of the experimental water (Pre) between the HW and PW groups. As shown in Table 4, there were no significant differences in the resting respiratory and circulatory parameters between the two groups. The continuous intakes of HW did not significantly change these parameters at rest. The peak values of VCO₂, RER, V_E, and HR were also not significantly changed after 2 weeks of intake of experimental water (Table 4).

The peak load was significantly elevated after the 2-week intake of experimental water, regardless of the experimental group (P < 0.01; Figure 4A). Importantly, the interaction tended to be significant (P = 0.067), suggesting that an increase in peak load from pre to post HW intake was potentially higher than that of PW intake. The difference in peak loads from before to after 2-week intake of experimental water also tended to be higher in the HW group than in the PW group (P = 0.075; Figure 4B). Because the body mass had changed over 2 weeks, we demonstrated VO_2peak divided by body mass (Figure 4C). VO_2peak of the PW group did not significantly change (P = 0.73), whereas that of the HW group was significantly elevated (P < 0.01). We also confirmed the presence of a significant interaction (water-by-time, P < 0.05; Figure 4C). In addition, the net increase in VO_2peak from before to after the 2-week treatment in the HW group was significantly higher than that in the PW group (P < 0.05; Figure 4D).

The exercise significantly increased the La levels, d-ROMs, and BAP in both groups, but HW did not significantly influence the response of these parameters to exercise (P > 0.10; Figure 5).

DISCUSSION

The major findings from the present study are as follows. First, no significant effects of HW on responses in blood La, oxidative stress, and antioxidant were observed. Second, a single intake of HW did not significantly increase peak load and VO_2peak2peak and tended to augment peak load. The present study suggests that continuous intake of HW enhances aerobic capacity in humans. Interestingly, similar results have also been reported in the previous meeting report.²³

Possible mechanisms underlying the improvement of aerobic capacity by continuous intake of HW

Although causation cannot be determined from our results, we can speculate the potential mechanisms that could underlie the increase in VO_2peak during an incremental cycling exercise test by continuous intake of HW. Maximal VO₂ (VO_2max) or VO_2peak is assumed to be mainly determined by 1) cardiopulmonary function that transports oxygen to the active muscle and 2) mitochondrial oxygen consumption (oxygen extraction and utilisation).²⁴²⁵²⁶ To the best of our knowledge, it has not been reported that continuous intake of HW improves cardiopulmonary function during exercise in healthy humans. Therefore, HW is unlikely to affect the oxygen supply system.

As for the latter determinant affecting VO_2max, i.e., mitochondrial function, mitochondrial reactive oxygen species have been suggested to impair mitochondrial activities.²⁷²⁸ Given that molecular H₂ has been suggested to directly and/or indirectly decrease oxidative stress,^29303132 it is possible that HW attenuated the decline in mitochondrial function evoked by exercise-induced oxidative stress.⁷ However, supplementation of HW did not reduce oxidative stress or increase antioxidant activity in this study. Therefore, it is logical to conclude that the H₂ scavenging reactive oxygen species mechanism did not operate in the present situation.

Recent studies have reported that HW enhances energy metabolism by inducing the expression of fibroblast growth factor 21 and/or peroxisome proliferator-activated receptor-γ coactivator-1α.³⁴ Moreover, Sobue et al.³³ proposed that H₂ can induce biological effects through the activation of a mitochondrial unfolded protein response via epigenetic histone modification and gene expression modification. Murakami et al.³¹ suggested that mild oxidative stress caused by H₂ enhanced mitochondrial oxidative phosphorylation. Taking together our findings and those of previous studies, we speculate that continuous intake of HW might increase mitochondrial energy production via the expression of these genes and proteins, thereby increasing the VO_2peak during incremental exercise.

In addition to the possibility that HW augmented mitochondrial energy production during the exercise, continuous intake of HW might have influenced mitochondrial biogenesis. The activation of adenosine monophosphate-activated protein kinase (AMPK) is known to facilitate mitochondrial biogenesis.³⁴ In fact, a previous investigation showed that the AMPK activator 5-amino-4-imidazolecarboxamide ribonucleoside (AICAR) augmented oxygen consumption rates and endurance capacity in mice without any physical training.³⁵ In addition, daily intake of AICAR increased the mitochondrial enzymes in skeletal muscle.³⁶ Toedebusch et al.³⁷ showed that continuous AICAR injections delayed the initial decline in lifetime-apex VO_2peak with ageing in rats. Importantly, HW could activate AMPK^{382peak by enhancing mitochondrial biogenesis through AMPK stimulation.}

In the present study, although continuous intake of HW significantly increased the VO_2peak, a single intake did not. The activation of mitochondrial metabolism and biogenesis are thought to require long-term administration of H₂,³⁴ or at least for more than a few hours.³¹³³³⁸ It is reasonable to presume that a single supplementation with HW was not enough to induce those effects.

Regardless of the experimental group, the peak load during incremental exercise in the current study was significantly elevated after 2 weeks of treatments. This result could be attributed to an adaptation to the repeatedly performed exercise test. Importantly, the increase in peak load from pre to post HW tended to be higher than that with PW. The augmentation in peak load could be due to the elevation in VO_2peak, namely HW-induced increase in oxidative energy metabolism.

It is interesting to speculate if the continuous intake of HW facilitated anaerobic metabolism during exercise, thereby increasing the peak load, as Aoki et al.¹¹ reported that supplementation by 1500 mL of HW suppressed exercise-induced La production. In the present study, the La response to exercise was not significantly different between the two groups, which was in accordance with a previous study,¹³ suggesting that HW did not affect anaerobic metabolism during the exercise, at least in the present situation. However, a further study is required, because the effects of the intake of HW on La response to exercise are still contradictory.¹¹¹³

Practical and clinical implications

H₂ has a safety advantage as it is not cytotoxic, even at high concentrations.¹ Drinking HW and inhalation of H₂ gas are two known administration methods.¹⁸ In the present study, we adopted HW instead of gas since it is more easily and safely administered, and thus more practical for use in daily life.¹⁸ It is well known that aerobic performance is strongly related to VO_2max or VO_2peak³⁹⁴⁰¹⁵ Cardiorespiratory fitness is an independent and strong predictor of all-cause and disease-specific mortality.⁴¹ Therefore, the present study showing that the continuous supplementation of HW enhanced aerobic capacity also implies that HW might contribute to maintaining and improving health.

Limitations

We acknowledge that the present study did not show any direct evidence to reveal the mechanism by which HW elevated VO_2peak. Additionally, the present study should be treated as a pilot study because we did not adopt a double-blind method when performing the experiments and did not determine the VO_2max. However, this study could be valuable as it showed the possibility that H₂ can be used as a supplement that enhances aerobic capacity in healthy humans.

Conclusion

The present study demonstrated that 2-week continuous supplementation of HW significantly augmented the VO_2peak and tended to increase the peak load in healthy individuals. These results suggest that continuous intake of HW potentially enhances the aerobic capacity.

Acknowledgements

We thank Ryota Masuda, Genki Ito, Kenji Funahashi, Keiichiro Kumagai, and Akiko Iino (Chubu University) for providing technical assistance.