PurposeExposure to environmental heat stress increases carbohydrate oxidation and extracellular heat shock protein 70 (HSP70) concentrations during endurance exercise at matched absolute, external work rates. However, a reduction in absolute work rate typically occurs when unacclimated endurance athletes train and/or compete in hot environments. We sought to determine the effect of environmental heat stress on carbohydrate oxidation rates and plasma HSP70 expression during exercise at matched heart rates (HR).MethodsTen endurance-trained, male cyclists performed two experimental trials in an acute, randomised, counterbalanced cross-over design. Each trial involved a 90-min bout of cycling exercise at 95% of the HR associated with the first ventilatory threshold in either 18 (TEMP) or 33 °C (HEAT), with ~ 60% relative humidity.ResultsMean power output (17 ± 11%,P< 0.001) and whole-body energy expenditure (14 ± 8%,P< 0.001) were significantly lower in HEAT. Whole-body carbohydrate oxidation rates were significantly lower in HEAT (19 ± 11%,P= 0.002), while fat oxidation rates were not different between-trials. The heat stress-induced reduction in carbohydrate oxidation was associated with the observed reduction in power output (r = 0.64, 95% CI, 0.01, 0.91,P= 0.05) and augmented sweat rates (r = 0.85, 95% CI, 0.49, 0.96,P= 0.002). Plasma HSP70 and adrenaline concentrations were not increased with exercise in either environment.ConclusionThese data contribute to our understanding of how moderate environmental heat stress is likely to influence substrate oxidation and plasma HSP70 expression in an ecologically-valid model of endurance exercise.