The purpose of the present study was to investigate the efficacy of MW as a rehydration beverage following moderate intensity cycling in a hyperthermic environment. It was hypothesized that ingestion of 1 L of MW following 2% BW loss would enhance physiological and perceptual markers of hydration, reduce fatigue, and enhance recovery over that of maple-flavored bottled water. Despite a higher osmolality and electrolyte content, the current study revealed no significant differences between MW and control on rehydration. However, likely due to differences in osmolality, thirst sensation remained elevated following consumption of MW, indicative of a potential for enhanced rehydration under ad libitum conditions. When sex differences were explored, on average, females had lower urine volume and output, resulting in tendency for greater ΔBW, but more concentrated urine, as indicated by higher UOsm, which taken alone could be suggestive of impaired rehydration. However, the greater ΔBW and lack of sex differences in the SOsm suggests a potential sex specificity in the mechanisms of rehydration, but further controlled work on these potential sex differences is warranted. Additionally, we observed differences in the kinetics between urinary and salivary measurements; salivary peaks occurred immediately post-exercise while urinary peaks were delayed at 0.5 h post-rehydration. Lastly, MW demonstrated a higher antioxidant potential (AP), which translated into elevated AP measured in the urine. Collectively, MW is a natural rehydration beverage with electrolytes and antioxidant potential but failed to enhance rehydration over water alone. Further work is needed to elucidate the sex differences, and temporal variations between biological compartments, during rehydration.
Rehydration: Carbohydrate-electrolyte sports beverages
Interventional studies on rehydration are relatively abundant in the literature [16,17,18,19,20,21], with the majority of these studies focusing on carbohydrate-electrolyte rich sports beverages, and the general consensus is that sports beverages are likely more effective in restoring sweat-induced hydromineral loss then water alone. However, the present study observed no differential effects of MW over water in terms of hydration (Fig. 2). Although MW boasts a rich electrolyte profile, the primary mineral-salts are Ca++, K+, carbonate, and sulfate, while Na+ is negligible (< 0.1 mg/L) (Table 2). However, as Na+ is a principle electrolyte lost in sweat; as compared to sodium-rich carbohydrate-electrolyte sports beverages (e.g. 457 mg/L of Na+) the relatively low Na + concentration in MW might explain, in part, the relative inadequacy in rehydration of MW and highlight that rehydration is likely sensitive to electrolyte composition. Though, previous work has suggested carbohydrate, protein, and/or caloric content might influence fluid uptake and rehydration [20, 58], perhaps due to relatively modest electrolytes, electrolyte composition, osmolality (~ 80 mOsm/kg), carbohydrates and caloric content (Table 2), we observed no such benefit of MW.
Nevertheless, arguments have been proposed against the efficacy of sports beverages. Shirreffs et al. investigated rehydration following intermittent exercise-induced dehydration (1.9% BW loss) in the heat . They observed attenuated mineral-salt balance, but not hydration status (% BW lost), with carbohydrate-electrolyte sports beverage. The authors speculated that salt-mineral replenishment may be inadequate with carbohydrate-rich electrolyte beverages, which is just as, if not more, important as hydration . Additionally, increased gastrointestinal (GI) discomfort following sports beverage consumption has also been observed [23, 28]. GI distress, likely due to individual variability in sensitivities to relatively high carbohydrate boluses post-exercise, as well as reduction of blood flow to the GI tract during exercise, which may blunt absorption, may explain the impaired mineral-salt balance observed in the previous studies . Furthermore, anecdotally there is apprehension as to the potential for excess chronic consumption of liquid calories as it relates to weight, and concerns regarding the use of artificial sweeteners and dyes, warrants further investigation into safer and natural alternatives.
Rehydration: Natural alternatives
Accordingly, there is a growing interest in natural, organic rehydration substitutes such as coconut water (CW) [27,28,29]. One study explored the potential impact of CW on rehydration after participants exercised at 65% of their VO2max at 32 °C and 53% RH, an experimental intervention similar to that of the present study . Rehydration was not significantly improved with CW. Similarly, when male volunteers exercised at 60% of their VO2max in the heat (31 °C, 51% RH) until 2.8% BW loss, %BW regained, rehydration index, blood volume, electrolyte profile, net fluid balance, and serum osmolality were not significantly different between CW and carbohydrate-electrolyte rich beverage , and agrees with other studies of CW [27,28,29]. Interestingly, in both studies, subjects were still hypohydrated by the end of testing, suggesting, perhaps that the study design could be optimized and/or CW might not be an effective rehydration choice.
The current study was the first to investigate the rehydration properties of MW. Using a similar experimental procedure, in accordance with previous studies investigating CW [27, 29], we found that consumption of MW was not different from water in rehydration (Fig. 2). Given an absolute volume (1 L), similar changes in ΔBW, UC (~ 2 units), USG (~.015), UOsm (~ 300 mOsm·kg− 1), and SOsm (~ 60 mOsm·kg− 1) were observed in both conditions, which returned to, or near to, baseline by the end of testing. However, despite intake of 1 L, MW sustained significantly greater perception of thirst over time (Fig. 4a), possibly due to the near six-fold greater osmolality concentration which might stimulate thirst and/or the modest amount of carbohydrates present in MW , but these differences in CHO content and osmolality were not great enough to elicit greater rehydration with MW. While not directly quantified in the present study, no participants reported any GI distress in response to the MW. Further, given the purported antioxidant properties of maple syrup , we sought to characterize the antioxidant capacity of MW, and revealed that MW had an antioxidant capacity ~ 4-fold higher than our control, and supports prior work which demonstrated, via the DPPH assay, that maple water has antioxidant capacity . The antioxidant capacity of MW, as determined by the FRAP assay, is on par with prune juice or grape juice . Although endogenous antioxidants (e.g. glutathione peroxidase) exist, exercise induces free radical outflow from muscle that may overwhelm our endogenous defenses . Analysis of urine antioxidant capacity, indicated that the acute exercise positively impacted antioxidant capacity, and MW enhanced this response at 0.5 h after ingestion of MW (Fig. 6). Finally, in contrast to our hypothesis, MW had no impact on fatigue (Fig. 4b).
Collectively, MW may be an accessible, and safer natural electrolyte-containing alternative to CW and sports drinks, particularly when consumed ad libitum. However, more work is needed to explore the impact of MW on hydration, such as larger populations, greater levels of dehydration, altered volume of intake (e.g. 120% of BW lost), measurement of plasma osmolality, and/or ingestion behavior (ad libitum vs. prescribed).
Rehydration: Potential sex effects
The current body of literature on the effects of sex on hydration is relatively limited. When matched for aerobic fitness and body fat percentage, Sawka et al.  found no changes in HR, rectal and skin temperature between males and females following treadmill exercise at 20 °C 40% RH, 35 °C 40% RH, and 35 °C 79% RH performed in both euhydrated and dehydrated states . Though, Eijsvogels et al. demonstrated in a relatively aged, and heterogeneous population that sex differences do exist in dehydration, which appear to be, at least in part, to differences in fluid intake . Moreover, focusing on rehydration specifically, work by Maughan et al. indicated that rehydration was not impacted by the menstrual cycle following exercise-induced dehydration to ~ 1.8%, a threshold similar to that of the present study . However, following exercise-induced dehydration, females in the current study had a significantly higher UOsm, and a trend for elevated USG (digital and analog), which remained elevated throughout the duration of testing (Fig. 2), which, taken alone, is suggestive of less effective rehydration. However, in the females, urine volume and cumulative output were significantly lower (Fig. 3), and ∆BW tended to be higher (Fig. 2), indicative of increased water retention and likely rehydration in women, though measurement of plasma osmolality would help discern possible sex differences in fluid shifts. These findings would agree with prior literature, where women are more likely to maintain plasma osmolality or develop hyponatremia due to fluid retention, then men who are more likely to become hypernatremic .
Given the limited research on the effects of sex on rehydration specifically, mechanisms behind these differences are not well understood. A tenable explanation is the hormonal fluctuations associated with the menstrual cycle or use of oral contraceptive pills , neither of which were controlled for the in present study, potentially obscuring the sex differences that are presently reported, and is limitation of the current study. Though, Maughan et al. found no effect of menstrual cycle phase in rehydration after exercise-induced dehydration , including the relatively low circulating hormone menstrual phase. Mechanistically, the variation in the hypothalamic-pituitary-gonadal axis over the menstrual cycle; specifically, estrogen and/or progesterone, may alter arginine vasopressin or its effect on the kidney, which ultimately influences hydration status [37, 38], and might explain the reduced, and concentrated, urine output (i.e. elevated USG and UOsm) in the females. Indeed, analysis of a subset of males and females urine for renin and aldosterone, revealed trends for greater renin in women, compared to men, but no such trend was observed in aldosterone. Importantly, if women were excluded and only the male data was analyzed the relatively minimal findings related to the rehydrating efficacy of maple water were unchanged, and thus the data remain combined. To our knowledge, our study might be the first to report sex differences in rehydration, using a relative work rate, and controlling for baseline hydration status, and based on our findings, there may be sex specificity in the mechanisms of rehydration. However, a limitation to the current study is that we did not control for menstrual cycle phase or OCP use, thus future studies are critically needed to specifically evaluate potential mechanisms of sex differences and potential influence of the menstrual cycle phase or oral contraceptive pill use impacts on rehydration, after exercise-induced dehydration.
Cardiovascular recovery from dehydration: Potential sex effects
The current study also investigated the potential impact of MW and/or sex on rehydration and measures of recovery using heart rate (HR) and HR variability (HRV). As expected, HR and HRV significantly increased and decreased, respectively following exercise-induced dehydration in the heat (Fig. 5) and returned to near baseline values for both conditions. These findings are somewhat in contrast to previous work by Carter et al., who demonstrated, using frequency domain measures, that HRV was impaired in recovery from dehydration and exercise in the heat .
When analyzed according to sex, RMSSD, a measure of parasympathetic contribution to HRV, significantly decreased following exercise (~ 120 pre vs. ~ 70 ms post) in both conditions, but returned to near baseline values for males only. A gap in the literature exists in regards to the potential role of sex on recovery of HRV, especially as it relates to heat- and exercise-induced dehydration. Therefore, it is not well understood the mechanisms behind the CV effects observed in present study. As mentioned before, menstrual cycle associated variation of sex (i.e. estrogen and progesterone) and non-sex hormones (arginine vasopressin) may likely explain these differences. Specifically, arginine vasopressin may increase sympathetic activity, which may attenuate recovery of HR and HRV , as observed in the present study. In support of this concept, in a subset of participants, urinary renin levels tended to be elevated in women, perhaps contributing to the impaired HRV. However, a limitation to the present findings is that menstrual cycle phase or oral contraceptive use were not controlled for, potentially obfuscating these reported sex differences. Mendonca et al. demonstrated sex differences in RR interval variability, but unlike the present study, they found that women, in response to a supramaximal Wingate test performed in normothermia, had enhanced cardiac parasympathetic input and a cardioprotective response (higher variability) . Future studies on the effects of sex on HRV measures, especially as it pertains to exercise in the heat and dehydration, and the potential role of menstrual cycle phase or OCP use are still needed.
Comparisons of hydration measures
To the best of our knowledge, no consensus exists about the optimal way to assess hydration status, thus a battery of hydration markers ought to be employed . Nude BW is a simple, quick, and practical assessment of hydration status, but is not without error or assumption . While blood borne assessments of hydration such as plasma osmolality (POsm), a relatively invasive measure, have been suggested to be a critical measure of static hydration and may not be sensitive to modest levels of dehydration [43, 44] due to highly controlled internal homeostatic mechanisms. On the other hand, urinary based measures, such as UOsm, are considered valid assessments sensitive to modest dehydration , but can be biased by fluid ingestion. SOsm has also been suggested to be a valid and effective method with similar sensitivity [9, 46], but also can be biased, short term by fluid ingestion.
Although not a primary research question in the current study, given the relatively comprehensive assessment of hydration we performed a preliminary examination of the methods. Incongruent with the urinary based measures of hydration suggesting adequate rehydration, the ΔBW suggested incomplete rehydration, due to diuresis, (Fig. 2), and agrees with prior work . Interestingly, while acute fluid ingestion is known to alter SOsm , the acute effect wanes 30 min post ingestion, thus, in the absence of further ingestion SOsm remained low, similar to urine, but still discordant to BW. Differences in kinetics between fluid compartments were also apparent. Specifically, salivary measures peaked immediately post-exercise, whereas urinary values had some latency peaking at 0.5 h (Fig. 2). Further, through using both analog and digital refractometers we found that the analog refractometer tended to overestimate USG, but tracked the digital refractometer almost exactly over time. To our knowledge we may be the first to report these findings, and such knowledge may be instrumental in the design of future studies, and/or applications where it may be desirable to perform rapid physiological determinations of hydration in clinical, field, or research settings.