Thermal comfort is an important aspect of the human health. When exposed to weightlessness, thermal comfort of the human body will be different from that in terrestrial condition, due to physiological changes induced by weightlessness. In order to study the human thermal comfort under weightless environment, a 3-stage experiment was conducted. In the first stage, 6 male subjects were exposed to 84 environmental conditions that were combined by 7 kinds of air temperature, 4 kinds of relative humidity and 3 kinds of air speed. During this stage the electrocardiogram (ECG) data of the subjects in sitting posture were recorded with ambulatory ECG recorders (Holter). Thereafter, ECG data were analyzed with the frequency-domain method, and the HRV indices were obtained. The second stage lasted for 7 days, during which the −6° head down bed rest (HDBR) model was selected to simulate physiological changes that usually appeared in real weightlessness. The third stage started after the 7-day HDBR, duringwhich ECG data were recorded and analyzed when all subjects were exposed to 84 environmental conditions and kept −6° HDBR. Among HRVindices, the LF/HF, representing the ratio of the low frequency power (LF) and high frequency power (HF) of the heart rate variability (HRV), was used to evaluate the thermal comfort. Then relationships between LF/HF and environmental parameters were scrutinized. Results showed that similar variations in LF/HF were observed for subjects before and after HDBR, when they experienced different air temperature, relative humidity and air speed. In addition, quadratic relationship was observed between the LF/HF and thermal sensation scores, and linear relationship was observed between the LF/HF and thermal comfort scores for subjects both before and after HDBR, meanwhile there were conspicuous differences in LF/HF between subjects before and after HDBR. Results revealed that thermal comfort of the human body under simulated weightlessness changed greatly compared with that under normal conditions, suggesting an alteration in thermoregulation.