The novel finding of this study is that a previously established pre-exercise water loading strategy using a combination of hydrating agents such as Cr and Gly that significantly increased body water compartments and reduced cardiovascular (Figure ) and thermoregulatory (Figure ) responses during running at 35°C, had no effect on the oxygen cost of running at 60% of

. The magnitude of change in BM following hyperhydration was similar to that previously reported in our laboratory [
19] and by Kern et al. (2001). Somewhat smaller differences in body water compartments were observed in the present study compared to the previous investigation by Easton et al. (2007). For example, Easton et al [
19] reported an increase of 0.9 L in TBW and 0.5 L in ICW after 7 days of supplementation. In the present study TBW and ICW were elevated by 0.7 and 0.3 L respectively after 7 days of supplementation. These differences could only be attributed to individual responses (i.e., level of "responders" to Cr supplementation as previously demonstrated) [
13,
34] as similar protocols were utilised. In the present study, the retained water was dispersed in both the ICW and ECW. Despite the significant increase in BM and body water compartments and consequently improved thermoregulatory responses during exercise, no significant differences in any of the respiratory variables were found between the pre- and post-supplementation exercise trials. Therefore, the finding that a significant increase in BM did not negatively impact on RE of trained runners supports the use of hyperhydration during endurance running when running in hot and humid conditions although confirmatory results are required during faster running speeds typical of sporting competition (i.e., > 85%

).
Temperature and cardiovascular regulation during exercise in the heat do appear to be critically dependent on hydration status [
35,
36]. In the present study, combined Cr and Gly supplementation induced significant hyperhydration and substantially attenuated the increase in HR at the end of the 30 min run at 35°C (Figure ). This attenuation of HR during exercise was of similar magnitude to that previous reported by Easton et al. (2007). As free water in the form of sweat is primarily lost from plasma and since no differences were found in PV changes pre- and post-supplementation despite the increase in TBW, ICW and ECW, it can be suggested that the increase in other water compartments resulted in water moving towards the plasma due to an osmotic gradient. This in turn leaves the PV unaffected. It should be also noted that in order for blood volume to be maintained in conditions of significant thermal stain and therefore sweating, fluid loss is obtained in varying proportions from ECW as well as ICW body water compartments [
37]. Furthermore, as loss of body water increases during exercise in the heat as a result of sweating, T
core also increases [
37]. Therefore, increasing body water could potentially result in better maintenance of T
core during exercise in the heat. Nose et al. [
38] reported a strong association between the loss of water in sweat and urine and the decrease in ICW after prolonged exercise in the heat. In the present study, Cr and Gly induced an increase in ICW and consequently, there was a significant attenuation in the rise of T
core during exercise in the heat (Figure ). It is possible that this Cr- and Gly-induced increase in ICW resulted in an increase of the specific heat capacity of the body [
13].
Published studies to date appear to confirm the reduction of T
core during exercise in the heat following Cr supplementation [
12,
13,
19]. Conversely, when Gly was used alone, ICW was increased without significantly attenuating the rise in T
core during the exercise period [
19]. The effects of Gly ingestion on T
core and thermoregulation in general during exercise in the heat is equivocal, with several studies reporting a reduction in T
core during exercise [
39] and numerous other studies finding no such effect [
16,
40]. In addition, several studies concluded that PV expansion has no effect on thermoregulatory responses or exercise performance during exercise in the heat [
9,
41]. These conflicting results and assertions provide strong support that the thermoregulatory benefits exhibited with Gly ingestion in the present study did not arise from any PV expansion but most likely from an increase heat capacity of the body. Nevertheless, it should also be noted that these thermoregulatory benefits were exerted when Gly was co-ingested with Cr.
Despite the significant increase in TBW and consequently improvement in cardiovascular and thermoregulatory responses during exercise, no differences in

were observed during running at 60%

. Coyle proposed that a reduction in BM induced by dehydration would impact on RE during marathon running by reducing the oxygen cost of running [
3]. In contrast, hyperhydration should theoretically increase the oxygen cost of running and therefore RE. However, no such effect was found in the present study. Furthermore, there was no increase in

over time during the trial at 10°C. The latter finding indicates that the subjects were working steadily at the calculated individual running speed corresponding 60% of

. It should be noted that this relatively low intensity was chosen in order to ensure that the present data would be comparable with previous studies conducted under similar conditions [
12]. Furthermore, the relatively low intensity was chosen as to secure that all subjects could complete the experiment in the heat while it was high enough to observe possible adaptations in cardiopulmonary or thermoregulatory parameters encountered with supplementation. However,

was increased during the trial in the heat. This was an expected effect as when exercising in hot environmental conditions, T
core rises accordingly. It has been shown that with an increase in T
core,

(and therefore RE) also increases [
42]. Despite this observation, no discernable difference in

between pre- and post-supplementation trials was reported. No other changes in any of the respiratory variables could be observed in the pre- and post-supplementation trials. Similar results have been reported in several other studies using Cr as the hyperhydrating agent [
13] as well as during constant load exercise in the study by Easton et al. (2007) where hyperhydration was induced by Cr and Gly [
19]. The data from the present study suggest that an increase in BM of approximately 1.4% (average increase in BM in the present study) has no significant effect on

. Whether such an increase in BM would influence running performance remains to be determined. Furthermore, as HR responses reflect those of

[
43], the finding that HR during exercise was not significantly different between pre- and post-supplementation trials conducted at 10°C is further evidence against any detrimental metabolic effect of the added BM induced by hyperhydration on RE.