The purpose of this study was to compare the effects of wearing different kinds of masks on the ear canal temperature, heart rate, clothing microclimate, and subjective perception of discomfort. Ten subjects performed intermittent exercise on a treadmill while wearing the protective masks in a climatic chamber controlled at an air temperature of 25°C and a relative humidity of 70%. Two types of mask—mask A, with exhaust valves and mask B, with exhaust holes—were used in the study. The results of this study indicated: (1) The subjects had a tendency toward lower maximum heart rate when wearing mask A than when wearing mask B. (2) Temperatures and absolute humidities (the outer surface of mask, the microclimate inside the mask, the chest wall skin and microclimate) of mask A were significantly lower than those of mask B. (3) The ear canal temperature increased significantly in mask B as compared to that in mask A. (4) The ear canal temperature showed significant augmentation along with increased temperature and humidity inside the mask microclimate. The mask microclimate temperature also affected significantly the chest microclimate temperature. (5) Mask A was rated significantly lower for perception of humidity, heat, breath resistance, tightness, unfitness, odor, fatigue, and offered less overall discomfort than mask B. (6) Subjective preference for mask A was higher. (7) The ratings of subjective overall discomfort showed significant augmentation along with increased wetness and fatigue. We discuss how the ventilation properties of masks A and B induce significantly different temperature and humidity in the microclimates of the masks and the heat loss of the body, which have profound influences on heart rate, thermal stress, and subjective perception of discomfort.