With the outbreak and spread of the COVID-19 epidemic, HCWs are frequently required to wear personal protective equipment (PPE) for nucleic acid sample collection in semi-open transition spaces. Wearing PPE causes significant psychological and physical stress in HCWs. In this study, operative temperature (Top) and wet-bulb globe temperature (WBGT) were used to assess thermal conditions through field experiments, while multiple physiological parameters were measured in the subjects. The results indicated that the subjects showed statistically significant differences in thermal perception and physiological parameters with and without PPE. Using observed increases in heart rate (HR), auditory canal temperature (Tac), mean skin temperature (MST), and end-tidal CO2 pressure, subjects were shown to have an increased metabolic rate and heat storage while wearing PPE. Additionally, a decrease in oxygen concentration was also observed, and this decrease may be linked to fatigue and cognitive impairment. Moreover, HR, MST, and Tac showed a significant linear relationship, which increased with temperature and operative temperature, and the HR response was stronger with PPE than without PPE. The neutral, preferred, and acceptable temperatures were significantly lower with PPE than without PPE, and the deviations for neutral Top/WBGT were 9.5/7.1 °C and preferred Top/WBGT was 2.2/4.0 °C, respectively. Moreover, the upper limits of acceptable WBGT, 29.4 °C with PPE and 20.4 °C without PPE, differed significantly between the two phases. Furthermore, the recorded physiological parameter responses and thermal perception responses of the subjects while wearing PPE indicated that they were at risk of thermal stress. Overall, these results suggest that people who wear PPE should focus on their health and thermal stress. This study provides a reference for the development of strategies to counteract heat stress and improve thermal comfort.