This is the first study to compare the thermoregulatory, cardiovascular and exercise performance effects during exercise in the heat induced by a known hyper hydrating supplement comprising of Cr/Gly and Glu [3, 4] and a newly designed supplement. The newly designed supplement differs from the already tested Cr/Gly/Glu, in the fact that part of the Glu is replaced by Ala. Ala is a compound characterized by the pronounced insulin-potentiating activity and thus known to potentiate Cr uptake under conditions when amount of carbohydrate added is significantly lower than the doses recommended for hyper hydrating supplement of Cr/Gly/Glu . The main finding of this study is that improvements in thermoregulatory and cardiovascular responses during exercise in the heat induced by Cr/Gly supplement containing excessive amounts of Glu and by Cr/Gly supplement containing Ala and lower amount of Glu are similar. We also found that exercise performance measured as time required to cover 16.1 km distance by cycling at 30.0°C and relative humidity of 70% was not improved following consumption of both supplements.
Ability of Cr/Gly/Glu and Cr/Gly/Glu Ala supplements to attenuate increase in Tcore and HR during exercise in the heat to a similar extent is not surprising, since in TBW increase in both groups was very similar and equal to 1.7 ± 1.1 and 1.2 ± 0.5 L in Cr/Gly/Glu and Cr/Gly/Glu Ala, respectively. The current study identified that following supplementation TBW was unchanged in 17% of participants; one from Cr/Gly/Gly group and two from Cr/Gly/Glu/Ala group. This most likely indicates that in these three participants Cr uptake was negligible and not sufficient for fluid retention in intracellular fluid compartments . Therefore these participants were considered as ‘non-responders’ and excluded from statistical analysis. This decision was made on previous suggestion that failure to discriminate between those who respond to Cr supplementation and those who do not could mask any effect resulting from Cr supplementation . No response to Cr supplementation by some participants is not surprising since muscle biopsies studies measuring Cr concentration before and after supplementation found that approximately 20–25% of the population show very little or no response to Cr supplementation . This can be explained by the fact that uptake of Cr by the skeletal muscle is very much dependent on initial Cr pool with uptake being highest in those with low levels . Although Cr uptake has been previously estimated with the measurement of urinary creatinine , this method requires the collection of urine for the duration of the supplementation period (7- days in the case of this study), which would be impractical, and too demanding for the participants of the current study. Previous study by Powers et al. (2003), which used muscle biopsy technique for the measurement of Cr uptake and D2O method for the measurement of TBW, has shown that increase in TBW was directly associated with Cr uptake .
In most previous studies examining the effects of Cr/Gly supplementation on hyper hydration, response to Cr/Gly supplement was determined by considering changes in BM rather than TBW changes [3, 4]. In our study both supplementation did not induce significant increase in BM, which is different to previous studies [3, 4]. It should be noted that changes in BM are influenced not only by hyper hydrating substances but also by changes in energy intake and energy expenditure during days of supplementation. In our study, during the week of supplementation energy intake including energy obtained from supplements was significantly lower. In addition some participants reported an ability to work harder in the training sessions during week of supplementation. Therefore, hyper hydration induced increase in TBW may not necessarily be reflected in BM. Gold standard technique such as D2O ingestion, for TBW measurements should be considered, since our study also demonstrated that correlation between TBW changes measured by D2O ingestion and estimated by BIA was not significant.
Another aspect related to the increase in TBW and is worth discussing, is the implication of TBW increase on PV. This was the first study to estimate impact of supplementation on pre exercise PV, via the direct measurement of tHb-mass with the use of the optimized CO-monoxide method . Both supplementations had no significant impact on PV although TBW increased by 0.2 – 4.6 L. We note that in our study estimated PV change following supplementation was small in relation to total PV and consisted of 28 mL and 132 mL in Cr/Gly/Glu and Cr/Gly/Glu/Ala groups, respectively, which is in accordance with suggestion of Latzka et al. (1998) . It is unlikely that a PV increase between 28–132 mL as occurred in the current study, accounts for the attenuation in the rise in Tcore and HR. Indeed, in studies where substantial alterations in cardiovascular function and heat storage by PV expansion were recorded, the magnitude of the PV changes was large (300–700 ml) [30–33].
Extend of supplementation induced attenuation of the increase in Tcore and HR during exercise seen in our study, is in consistency with previous studies [3, 4]. Rise in Tcore was reduced by 0.2 and 0.3°C following Cr/Gly/Glu and Cr/Gly/Glu/Ala supplementation respectively (Figure 4). Hyper hydration achieved through Cr/Gly/Glu and Cr/Gly/Glu/Ala supplementation in the present study was also successful in attenuating the increase in HR by up to 2 and 4 beats/min respectively, during the constant load exercise in the heat (Figure 3). This was achieved regardless of the fact that changes in PV during exercise conducted before and following supplementations were not significantly different. Therefore, it seems that improvement in thermoregulation induced by hyper hydration strategies used in this study were achieved by PV and sweat rate maintenance  and by increasing the specific heat capacity of the body as suggested by Easton et al. (2007) and Beis et al. (2011), rather than PV expansion.
We found that in Cr/Gly/Glu group, following supplementation, RER during constant load exercise was significantly higher than in the pre supplementation trial which reflects the contribution of CHO towards energy production being enhanced and contribution of fat reduced by consumption of the Cr/Gly/Glu supplement. This finding is not surprising since daily amount of Glu consumed with the Cr/Gly/Glu supplement for the duration of seven days was as high as 150 g and significantly increased intake of available CHO. It is well established that increased dietary carbohydrate intake for several days increases muscle glycogen concentration [35, 36] and that energy substrate selection during exercise to a great degree depends on muscle glycogen availability [37, 38]. In Cr/Gly/Glu/Ala group, RER values measured during constant load exercise were not significantly different between pre and post supplementation trials. This can be explained by lower intake of Glu within the Cr/Gly/Glu/Ala supplement in comparison to the Glu contained in the Cr/Gly/Glu supplement. Regardless of the possible enhanced availability of muscle glycogen and change in energy substrate utilization during exercise following Cr/Gly/Glu suplement, it is unlikely that this could have impact on exercise performance due to muscle glycogen depletion. This suggestion receives support from no hypoglycemia being sees at point of completion of all time trials.
Despite the decrease in Tcore and HR during constant load exercise experienced by both supplementation groups in the present study, time trial performance was not affected which is in consistency with some hyper hydration studies [3, 39, 40] but contradict findings of other researchers [5, 41–43]. It should be noted, that studies finding a positive effect of hyper hydration on exercise performance, employed protocols different from that in our study. For example, in the study by Anderson et al. (2001), participants were required to cycle for 90 min at a constant load before commencing the time trial. This duration is more than twice the duration employed in the current study. In addition, it might be that in our study, intensity of constant load exercise has not been high enough since mean values of RPE were 15 and 14 in Cr/Gly/Glu and Cr/Gly/Glu/Ala group, respectively (Figure 5). It is therefore possible, that the exercise trial in the present study was not of sufficient duration and intensity for hyper hydration to have a significant effect on performance. On the other hand, values for mean O2 during constant load exercise were 44 and 42 ml/kg/min in Cr/Gly/Glu and Cr/Gly/Glu/Ala group, respectively, which consisted of ~ 70% of O2max (Table 1) and should be high enough to induce fatigue prior to the time trial. It is therefore possible that commencing exercise in a hyper hydrated state might not confer any significant advantage in terms of exercise performance as found in the studies by Easton et al. (2007), Marino et al. (2003), and Latzka et al. (2000). In either case, studies with duration and conditions sufficient to induce a higher degree of dehydration should be carried out to examine whether hyper hydration can have a significant effect on exercise performance.