During dehydration fluid moves from the plasma to both intracellular and extracellular spaces and then eventually back to the circulation 12. Pressure changes involving hydrostatic, oncotic and osmotic forces control the dynamics of fluid movement 1. This has important implications for thermoregulation and athletic performance. Significant performance decrements have been shown with hypohydration levels of only 2% 3. Considering that a thirst sensation may not develop until this level of hypohydration has already been reached, it becomes critical for athletes to rehydrate before they feel the need to drink.

Several sport drinks are marketed to be a more effective means of promoting rehydration and maintaining exercise performance than water alone. However, little research is available to support the efficacy of these drinks during relatively short duration endurance exercise (≤ 2 hr). Water appears as effective as any sports drink during exercise in maintaining performance and thermoregulation 4. Interestingly, recent advances in sport supplements suggest the use of certain organic osmolytes such as glycine betaine may provide some protection of intracellular fluid volume 5; however, its ability to affect performance is not clear.

Recent research demonstrates that certain amino acids may also promote rehydration by enhancing water and electrolyte absorption 6. Glutamine has been reported to increase electrolyte and water absorption in both animal and human subjects suffering from intestinal infections 789, but not in others 10. However, differences may be related to the stability issues related to glutamine. Fürst 11 suggested that glutamine derivatives such as alanyl-glutamine may be more stable than glutamine by itself, especially at low pH. This could be a potential scenario during exercise when increases in lactic acid are common. Lima and colleagues 6 reported that alanine and glutamine together is more stable than glutamine alone in increasing electrolyte and water absorption, likely via an improvement in ion transporters within intestinal epithelia.

Both glutamine and alanine/glutamine in combination have been shown to be effective for antioxidant defense during situations of severe illness 121314. In addition, glutamine has been shown to be an effective modulator of the immune response to exercise 15 and possibly improve athletic performance 16. However, there is considerable debate in this area 17, which justifies further investigation. Thus, the purpose of this study was to examine the efficacy of two different doses (0.2 g·kg^-1 and 0.05 g·kg^-1) of the dipeptide L-Alanyl-L-Glutamine on performance, recovery and the fluid regulatory response during an exhaustive endurance exercise protocol following a 2.5% dehydration stress. In addition, the effect of this dipeptide L-Alanyl-L-Glutamine on endocrine and biochemical markers of inflammation, oxidative stress and immune response during the exercise and dehydration stress was also examined.

U_sg (1.026 ± 0.004), U_osm (813 ± 299 mOsm) and P_osm (297.0 ± 4.6 mOsm) were similar for all trials at DHY. These results reflected the overnight fasting and exercise-induced dehydration performed during prior to each trial. Plasma glutamine concentrations were significantly higher for all groups at RHY and IP compared to BL (p = 0.002 and p = 0.000, respectively) and DHY (p = 0.001 and p = 0.000, respectively) (Figure 1). [Glutamine] for T5 were significantly higher at RHY and IP than T2 - T4. AUC analysis showed significantly greater [glutamine] for T5 at all time points compared to the other experimental trials (see Figure 2).

Time to exhaustion was significantly reduced during T2 than at any other experimental trial (see Figure 3). When examining Δ performance (difference between each experimental trial and T1), time to exhaustion was significantly greater during T4 (130.2 ± 340.2 sec) and T5 (157.4 ± 263.1 sec) compared to T2 (455.6 ± 245.0 sec, p = 0.05 and p = 0.01, respectively) (Figure 4). No other between trial differences were noted. Cardiovascular changes during exercise are depicted in Table 1. No significant differences in either resting or post-exercise HR occurred between trials. In addition, no differences occurred in resting BP between trials, however, systolic BP post-exercise was significantly lower at T2 and T3 compared to T1. No other differences existed in systolic or diastolic BP response between trials. No changes in RER occurred between trials.

There were significant main effects for both La^- (p = 0.000) and GLU (p = 0.000) responses to the exercise protocol (Table 2). There were also significant elevations at IP in both of these variables compared to all other time points. However, there were no significant differences between trials. A main effect for time (p = 0.011) also occurred for plasma osmolality. P_osm at IP (300.4 ± 16.7 mOsm) was significantly elevated compared to BL (295.0 ± 3.9 mOsm, p = 0.010) and RHY (293.9 ± 4.9 mOsm, p = 0.002) but, not DHY (297.0 ± 4.5 mOsm, p = 0.100). No other significant differences were noted. In addition, no between trial differences in P_osm were observed. A significant main effect for time (p = 0.001) was also observed for plasma potassium concentrations. Plasma potassium was significantly elevated at IP compared to BL (p = 000), DHY (p = 0.000) and RHY (p = 0.017). No other differences were noted and no between trial effects were observed. A significant main effect for time (p = 0.000) was also observed for plasma sodium. Plasma sodium concentrations at IP and DHY were significantly greater than that observed at BL (p = 0.000 and p = 0.000, respectively) and RHY (p = 0.000 and p = 0.000, respectively). When collapsed across time, plasma sodium concentrations were significantly greater at T2 than compared to all other experimental conditions. Plasma sodium concentrations were also significantly greater for T2 than all other experimental trials at RHY (p = 0.000) and IP (p = 0.000). AUC analysis also demonstrated a significantly greater sodium concentration for T2 compared to all other trials.

The ALD response to the experimental trials is presented in Figure 5. A significant main effect for time (p = 0.013) was observed. [ALD] at RHY and IP were significantly lower than that at BL and DHY (Figure 5). No other significant differences were noted and no significant interactions were observed. The plasma AVP responses are shown in Figure 6. A significant main effect for time (p = 0.000) was also observed. AVP was significantly elevated at DHY (p = 0.000), RHY (p = 0.000) and IP (p = 0.000) compared to BL measures. In addition, AVP concentrations at DHY were significantly higher (p = 0.05) than IP across all trials. There were no significant differences between trials, and no significant interactions between time and trial.

No significant differences were observed between trials in CRP, IL-6, and MDA response to the exercise and hydration stress (see Figures 7, 8 and 9, respectively). A significant main effect for time was observed for both CRP (p = 0.000) and MDA (p = 0.000). BL concentrations for both of these variables were significantly lower than all other time points. There was a significant main effect for trial for MDA between T3 and T5 versus T2 (p = 0.004 and p = 0.008, respectively) and T4 (p = 0.05 and p = 0.011, respectively). Evaluation of the response of IL-6 revealed a significant main effect for time (p = 0.000). IL-6 concentrations were significantly greater at IP than at BL (p = 0.000), DHY (p = 0.000), and IP (p = 0.000). In addition, IL-6 concentrations at RHY were significantly higher than at BL (p = 0.000) and 24P (p = 0.006). AUC analysis for CRP, IL-6 and MDA did not reveal any significant differences between trials.

No significant differences from BL were seen in the testosterone response to the exercise and dehydration stress during any experimental trial (Figure 10). A significant main effect for time was seen in both the ACTH (p = 0.000) and cortisol (p = 0.000) response to the exercise and dehydration protocol (Figure 11 and 12, respectively). When collapsed across trials, significant elevations in cortisol and ACTH concentrations were seen at IP and 24P compared to BL, DHY and RHY. No other significant differences were noted and no between trial effects were observed. A significant main effect for time (p = 0.000) was seen in the growth hormone response. When collapsed across trials, growth hormone concentrations were significantly elevated at IP compared to all other time points (Figure 13). No other differences were observed. AUC analyses for testosterone, ACTH, cortisol and growth hormone did not result in any significant differences between trials. No significant difference from baseline concentrations (43.9 ± 18.7 IU) was seen in creatine kinase concentrations during any trial.

Plasma volumes decreased -5.45 ± 11.38% at DHY for all experimental trials, plasma volumes were decreased at RHY (-6.78 ± 11.27%) for all experimental trials and continued to decrease at IP (-21.44 ± 10.54%). However, the differences between trials were not significant. Blood variables were not corrected for plasma volume shifts due to the importance of molar exposure at the tissue receptor level.

The results of this study showed that when subjects are hypohydrated by 2.5% of their body mass and exercise to exhaustion, significant performance decrements occurred. However, when subjects ingested the AG supplement during the rehydration period (T4 and T5) the magnitude of performance decrement was significantly less compared to the dehydrated condition (T2). Water alone (T3) did not appear to significantly reduce the performance decrement. Despite significant performance improvements for both T4 and T5, glutamine concentrations were significantly elevated at only T5 for both RHY and IP compared to all other trials. As expected, the higher dose of AG produced a greater increase in plasma glutamine concentrations. The time course of glutamine appearance in plasma is similar to that reported by Klassen and colleagues 20. In that study, a 20 g oral feeding (approximate to the high dose [T5] used in this study) resulted in a peak increase occurring at 49 ± 8 min (range 30 - 120 min) following dosing, which corresponded to the RHY and IP blood draws. Although dosing patterns of 0.1 g·kg·BM^-1 can increase plasma glutamine concentration by approximately 50% 21, the ability to increase plasma glutamine concentrations with doses lower than 0.1 g·kg·BM^-1 is not clear. Based on the present findings a dose of 0.05 g·kg·BM^-1 AG did not result in a significant elevation in plasma glutamine concentrations.

Despite the lack of any significant increase in plasma glutamine concentrations at T4, significant performance improvements were found for both T4 and T5. It is possible that in instances where plasma glutamine concentrations are normal, small bolus samples may be sufficient to offset mild hydration perturbations. The AG dipeptide has an important role in fluid and electrolyte uptake in the gut. AG appears to increase electrolyte and fluid uptake across the intestines by increasing ion transport through an enhanced signaling pathway within the intestinal mucosal cells 622. Further, AG supplementation has also been demonstrated to enhance muscle glutamine uptake 23. Although speculative, it is likely that an enhanced glutamine uptake by skeletal muscle will also result in a greater sodium uptake, which is supported by the reduced sodium concentrations at T4 - T5 compared to T2. The enhanced sodium uptake by skeletal muscle may have contributed to a reduction in fatigue by maintaining strength and efficiency of muscle contractility 24. In addition, although plasma glucose concentrations were not different between trials, alanine is a gluconeogenic substrate and may have contributed to the delay in fatigue by sparing muscle glycogen 2526.

ALD responses were significantly lower at RHY and IP for all trials, with no between trials differences observed. Although ALD is reported to respond in a graded manner to levels of hypohydration 2728, the magnitude of hypohydration in this study was likely not sufficient to stimulate increased ALD production, and rehydration likely resulted in the significant decline of ALD across trials at RHY and IP. These findings agree with observations that ALD concentrations will decline when water or electrolyte drinks are provided during exercise 29. The similarity in the ALD response found in this study may also be attributed to similar plasma volume changes observed between trials 29. In addition, ALD is also stimulated by changes in BP 30. In this present study, there was a significantly lower BP at IP during the T2 and T3 trials compared to T1. This most likely reflected a greater local fatigue resulting from the hydration perturbation contributing to the reduced time to exhaustion compared to T4 and T5 (an approximate 20 mmHg difference [p > 0.05] was found between post-exercise systolic BP at T2, T3 compared to T4 and T5). The significantly lower ALD responses at RHY and IP for all trials likely reflect the lack of a strong single stimulus (e.g. level of hypohydration) and represent the multitude of physiological factors that influence ALD secretion.

Our findings also indicated that AVP was significantly elevated from BL at all time points and that AVP concentrations at IP were significantly greater than DHY as well. However, the AG supplement was unable to alter the response of AVP to this mild dehydration and exercise protocol. The response to the exercise protocol was consistent with previous studies examining a similar exercise intensity 28. Changes in AVP concentrations are dependent upon exercise intensity and changes in P_osm and blood volume 3132; thus it is not surprising to see no significant differences between the trials in the AVP response considering that no between trial differences were noted in P_osm or plasma volume changes. The mild dehydration and exercise protocol was unable to create any difference to the fluid regulatory response between the various trials. Previous studies examining the effect of hypohydration levels have typically examined body water deficits of greater magnitudes (~5%) and greater differentials than that used in this present study 2829.

CRP is often used as a marker of inflammation and muscle damage 333435. Previous studies have shown that CRP will increase in response to prolonged physical activity such as triathlons 35 and marathons 33 but not during shorter duration exercise 3436. It is likely that the relatively short duration in time to exhaustion, despite the added mild dehydration stress did not cause a significant inflammatory response. Many studies use CK as a marker for muscle damage and have suggested that a rapid acute phase inflammatory response (reflected by an increase in CRP within 24 hours post-exercise during eccentric exercise in untrained individuals) can initiate delayed onset of muscle soreness and additional tissue necrosis occurring following 24 hours post-exercise is reflected by elevations in CK 37. Although CRP concentrations observed in this study were significantly elevated from baseline levels, they were not different between DHY, RHY, IP and 24P suggesting that any changes may have been the result of plasma volume shifts and not due to an inflammatory response. This is supported by the response of CK during each trial (no change from baseline concentrations). Interestingly, the AG supplement was unable to provide any indication for an attenuation of the inflammatory or muscle damage response to the exercise protocol. This is likely due to the limited inflammatory response and lack of a clear indicator of muscle damage as measured by CK.

There was a significant elevation in serum concentrations of IL-6 at IP compared to BL, DHY and 24P and at RHY compared to BL and 24P. This response is consistent with previous studies that have shown significant elevations following prolonged endurance 333538 and eccentric exercise 34. IL-6 is produced in active skeletal tissue 39 and in the central nervous system 40. Exercise is a potent stimulator of IL-6 production, with elevations greater than 100-fold reported 41. It is thought that increases in IL-6 modulates CRP production in the liver 42 and operate synergistically to enhance the inflammatory response to exercise. The potential outcome from this inflammatory response is the risk for significant tissue damage and reduced recovery capability.

Several investigations have examined the ability of nutritional intervention to attenuate the post-exercise inflammatory response 4344. Carbohydrate ingestion 44 and a vitamin E and omega-3 fatty acid combination 43 have been successful in attenuating the IL-6 response to exercise. In contrast, glutamine supplementation has been shown to enhance plasma IL-6 production 38, while an AG dipepide has shown to have no effect on cytokine production in healthy individuals 45. Hiscock and colleagues 38 suggested that the enhanced glutamine uptake by skeletal muscle would increase or maintain the production of IL-6. This hypothesis may be more consistent with the anti-inflammatory role suggested of IL-6 during exercise 46. Increases in IL-6 concentrations have been consistently reported without corresponding muscle damage 46, and is supported by the results of this present study. The difference between this study and the results of Hiscock et al., 38 may be related to the length of exercise and the training experience of the subjects. In the present study the duration of exercise ranged from 5 - 47 minutes following the ~60 minute active dehydration protocol, in recreationally trained individuals, while the subjects in Hiscock's study were untrained and required to perform a 2-hr time trial using the same exercise intensity as employed in this study. However, those subjects were euhydrated and allowed to drink ad libitum. It is unlikely that dosing impacted these results, considering that the glutamine dose used in Hiscock's study (3.5 g) was similar to the low dosing trial (T4).

[MDA] were significantly elevated from baseline for all trials. This is not surprising considering that exercise is a potent stimulator of the formation of reactive oxygen species 47. The results of this study are also consistent with previous research demonstrating elevated oxidative stress following a mild dehydration and exercise to exhaustion protocol 48. However, in contrast to Paik and colleagues 48 rehydration with water, or water and the supplement, was unable to reduce the MDA response to the exercise protocol. It is likely that the differences between these two studies may reflect the methods used to achieve dehydration. Paik et al., 48 used passive means in the heat (sauna exposure) to achieve 3% hypohydration, while this present study used both a passive and active (exercise) dehydration protocol to achieve the 2.5% body weight loss. Although speculative, it is possible that differences between methods used for dehydration may have resulted in a different oxidative stress. The time used to achieve body weight loss, although performed at a lower intensity of exercise, resulted in significant elevations in MDA concentrations that were not altered by water or water and AG.

The anabolic and catabolic response to the study protocol did not differ among trials suggesting that the supplement was unable to provide any significant benefit regarding enhanced recovery from the exercise and hypohydration stress. It is also possible that these hormonal measures may not have been sensitive enough for assessing recovery from a moderate dehydration and endurance exercise protocol 49. [TEST] did not significantly elevate from baseline levels following exercise despite a reduction in plasma volume. This is not surprising considering that subjects experienced only a moderate hypohydration stress and that time to exhaustion ranged from 13 - 18 minutes. Exercise of relatively short duration (i.e. 10-20 minutes) does not appear to increase [TEST] 5051, even with a mild hydration perturbation in fit individuals 52.

The CORT response was consistent with previous studies that have shown that hydration levels do not influence [CORT] 5253. The post-exercise elevation in CORT was also consistent with the metabolic stress associated with moderate exercise and hypohydration 5354. Results of this study though were unable to show that CORT responses can differentiate between levels of hypohydration, which contrasts with observations made by Judelson et al., 55 and Maresh et al., 54. However, the ability for hypohydration to modify the catabolic response to exercise appears to be more relevant when hypohydration reaches 5% or greater, or when exercise is performed at higher exercise intensities 5455. These findings also suggest that the pituitary-adrenal axis responds similarly to this exercise and hypohydration perturbation as ACTH responded in a similar pattern as CORT, with no influence from the AG supplementation.

GH secretion patterns have been shown to be quite responsive to changes in the acid-base balance of muscle 56. Considering that no differences were noted in the La^- response between the trials, the GH response to the exercise and hypohydration stress appears to have responded in a normal manner. These results are also in agreement with Judelson et al., 55 but, contrast with Peyreigne and colleagues 57. In this latter study, it was suggested that a hydration stress could blunt the GH response to exercise. Interestingly, there was an 18% and 20% greater GH response (p > 0.05) during T3 and T4 versus T2, respectively, while a 42% difference was found between T5 and T2 (p > 0.05). Although these responses were not significantly different, it does suggest an interesting trend that provided some support to previous results 57. Whether the high dose glutamine ingestion played a role in this response is not clear. Previous investigations have suggested that glutamine concentrations can elevate the GH response at rest 5859, but not exercise 58. It appears that the most compelling stimulus for glutamine's role in stimulating GH release is during prolonged critical illness when plasma glutamine concentrations are below normal levels 60. Thus, the high variability in the GH response in this study may be attributed to the normal glutamine concentrations at rest, however the largest gains in GH occurred during the trial (T5) that glutamine concentrations were significantly higher than T2 - T4.

In conclusion, the results of this study demonstrate that AG supplementation provides significant ergogenic benefits by increasing time to exhaustion during a mild hydration stress. This ergogenic effect was likely mediated by an enhanced fluid and electrolyte uptake. AG supplementation, irrespective of dosing, did not have any effect on immune, inflammatory or oxidative stress responses. Results also indicated that the AG supplement did not influence the pituitary-adrenal-testicular axis during this exercise and mild hypohydration perturbation.

Kyowa Hakko USA (New York, NY) provided funding to The College of New Jersey for this project. All researchers involved independently collected, analyzed, and interpreted the results from this study and have no financial interests concerning the outcome of this investigation. Publication of these findings should not be viewed as endorsement by the investigator, The College of New Jersey or the editorial board of the Journal of International Society of Sports Nutrition.

JRH was the primary investigator, obtained grant funds for project, designed study, supervised all study recruitment, data/specimen analysis, statistical analysis and manuscript preparation. NAR, JK, SLR, NK, AMG, MS, SA, and ADF oversaw all aspects of study including recruitment, data/specimen analysis, and manuscript preparation. BLB, LMY, LLH, BK and CMM were co-authors, assisting with data analysis. All authors have read and approved the final manuscript.