Objective: Hot environments are associated with impaired glucose metabolism at rest in healthy humans. The purpose of this study was to explore the contribution of key glucoregulatory hormones and biomarkers to this altered glucose tolerance. Methods: The effects of ambient temperature on glucose tolerance and its determinants were assessed with a 3-hr oral glucose tolerance test (OGTT) administered to 19 healthy young men and women at 22 °C and 31 °C. Results: The glucose response amplitude was greater in warm environment (AUC 904 ± 151 vs. 721 ± 89 mmol/l·180 min at 31 °C and 22 °C, respectively, p < .001). There was no significant effect of environmental temperature on insulin, growth hormone or pancreatic polypeptide concentrations (all p > .17). The cortisol response to the glucose load was reduced 30, 60, 90 and 120 minutes postload at 31 °C compared with 22 °C (p = .001). The interleukin-6 concentration was also lower in the session at 31 °C (p = .043). Conclusion: We conclude that the effects of environmental temperature on the glucoregulatory hormones and biomarkers reported in this study do not explain the exaggerated increase in blood glucose after a glucose load taken in a warm environmental temperature.