The purpose of this study was to clarify whether hot pack therapy can change the blood flow of human masseter muscles. Thirty-two healthy subjects with no history of muscle pain in the masticatory system participated and were divided into two groups. One group underwent proper hot pack therapy (hot pack group) and the other underwent sham hot pack therapy (control group). Continuous and non-invasive measurements of haemoglobin volumes and oxygen saturation levels (StO2) were determined with a near-infrared spectroscope. The blood flow parameters were total haemoglobin volume (THb), oxygenated haemoglobin volume (OXHb), deoxygenated haemoglobin volume (deOXHb) and oxygen saturation level (StO2). In hot pack group, results showed that the THb, OXHb and StO2 after the hot pack application were significantly larger than those before the hot pack. In control group, the THb, OXHb, deOXHb, StO2 and heart rates showed no significant differences between the values before and after the sham hot pack application. The THb, OXHb and StO2 after the hot pack application in hot pack group were significantly larger than those in control group, while the deOXHb after the hot pack was significantly smaller than that in control group. The heart rates showed no significant differences between the groups. The results suggest that hot pack therapy can increase regional blood flow of human masseter muscles and creates an advantageous condition for aerobic energy metabolism in the muscles.