This study investigates the varies in human physiological response, subjective sensation and acute subclinical health symptoms with increasing activity levels at different high temperatures. Thirty-two healthy subjects were recruited to walk on a treadmill in a climate chamber at a speed of 4 km/h. They experienced four temperature conditions (26 degrees C, 30 degrees C, 33 degrees C and 37 degrees C), each exposure lasting 85 min. Eardrum temperature, heart rate, skin temperature, systolic blood pressure, respiratory flow, and respiration rate changed significantly with increasing temperatures. At temperature of 37 degrees C, the SpO2 decreased significantly compared with at 33 degrees C. Subjects perceived the environment unacceptable at 37 degrees C. The perceived air quality and air freshness correlated linearly with the enthalpy of air. The intensity of headache, dizziness, fatigue and sleepiness increased with increasing temperatures, while only aggravated significantly at 37 degrees C. Additionally, compared with the results at light activity level, heart rate and other key physiological parameters increased significantly with increasing activity levels. The subjects felt "very hot" at 37 degrees C, and the change trend in symptoms reported by subjects increased significantly at 37 degrees C with the increased exposure time, while no significant change was observed in 26-33 degrees C. It indicates that exposure to 37 degrees C impairs the health and safety of heat acclimatized subjects. Using linear fitting curve to predict human physiological tolerance time suggested by ISO 9986. The result shows that eardrum temperature exceeded 38.5 degrees C for 97min continuously walk at 37 degrees C. This provides valuable information involved physiological and psychological responses when human exposed to high temperatures in daily life or industrial production.