Endurance exercise combined with environmental heat stress alters substrate metabolism and upregulates extracellular heat shock protein 70 (HSP70) concentrations. Previous research has studied these stimulatory effects of heat stress at matched absolute, external work rates. In the real-world, however, a reduction in absolute work rate typically occurs when training and/or competing in hot environments compared to temperate conditions. The aim of the present investigation was to determine how heat stress impacts substrate oxidation rates and plasma HSP70 expression during endurance exercise regulated by relative physiological work rates, or heart rates (HR). Ten endurance-trained, male cyclists performed two experimental trials in an acute, randomised, counterbalanced cross-over design. Each trial involved a 90-minute bout of cycling exercise at a specified HR (95% of the HR associated with VT1) in either 18 (TEMP) or 33 ̊C (HEAT), with ~60% relative humidity. Mean power output (17 ± 11%, P = 0.0005) and whole-body energy expenditure (14 ± 8%, P = 0.0003) were significantly lower in HEAT. Sweat rate was significantly greater in HEAT (87 ± 63%, P = 0.0003). Whole-body carbohydrate oxidation rates were significantly lower in HEAT (19 ± 11%, P = 0.002), while fat oxidation rates were not different between-trials. Plasma HSP70 expression was not increased with exercise in either environment. 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), while the lack of plasma HSP70 accumulation during endurance exercise may be explained by insufficient exercise duration, inadequate thermal stress, and/or the cycling-exercise modality employed in this present study. These 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.