Several reports have suggested that the risk of exertional heat illness (EHI) in Thoroughbred racehorses increases in high ambient temperatures. Heat dissipation in horses during exercise becomes less efficient when the body temperature and ambient temperature are close. Therefore, we hypothesised that exercise at 40 degrees C may increase body temperature, oxygen consumption, and cardiac output during incremental exercise tests compared to 20 and 30 degrees C. Six trained Thoroughbred horses were studied in a randomised, crossover design at three ambient temperatures with a 6-day washout period. Using a 3% inclined treadmill, horses performed incremental exercise tests at 1.7, 3.5, 6, 8, and 10 m/s for 90 s at ambient temperatures of 20, 30, and 40 degrees C. The effects of ambient temperature at 10 m/s on physiological variables were analysed using mixed models (P<0.05). Pulmonary arterial temperature and rectal temperature at 40 degrees C were higher than those at 20 degrees C (P<0.001) and 30 degrees C (P<0.001). Similarly, oxygen consumption (vs 20 degrees C, P=0.009; vs 30 degrees C, P=0.006) and cardiac output (vs 20 degrees C, P=0.001; vs 30 degrees C, P=0.001) at 40 degrees C were higher than those at 20 and 30 degrees C. Arterial O-2 partial pressure, O-2 saturation, and pH at 40 degrees C were lower than those at 20 and 30 degrees C. Arterial CO2 partial pressure at 40 degrees C was higher than that at 20 and 30 degrees C. No differences were observed in arterial-mixed venous O-2 concentration difference (P=0.391) and plasma lactate concentration (P=0.134) at different ambient temperatures. These results indicate that exercise at 40 degrees C causes excessive high body temperature, decreased running economy, and increased cardiac output compared to exercise at 20 and 30 degrees C. We strongly suggest that trainers and veterinarians should anticipate the occurrence of increased thermal stresses when ambient temperature is extremely high even in dry conditions and prepare to mitigate the risk of EHI from the perspective of equine welfare.