This study investigated the physiological thermal response of people under simulated weightlessness using a 6° head down bed rest (HDBR) model to ensure the human body functions in the weightlessness way. Thermal responses of six male participants were experimentally determined. The relationship between thermal comfort and heart rate variability (HRV) was also scrutinized. Physiological responses of participants under different environmental conditions were observed including core temperature, sweating, and most importantly, HRV. Elevated core temperatures were observed amongst participants, with a maximum increment of 0.5°C. The sweating of participants under simulated weightlessness was conspicuously retarded, requiring a 2–4°C increase in environmental temperature to start sweating. Body regional differences in the sweating were also observed. The HRV index ‘LF/HF’, which reflects the balance of the autonomous nervous system, was found to vary with the environment and closely associated with the variation in the thermal comfort scores. Participants under simulated weightlessness had higher mean LF/HF when they felt comfortable, and they preferred a warmer environment in comparison to under normal gravity condition. An altered thermoregulation of the human body under simulated weightlessness was indicated by our findings. The use of HRV index could provide a useful prediction of subjective thermal comfort under weightlessness.