This study aimed to investigate the acute effects of stretching exercise on skin temperature, flexibility, passive muscle stiffness, and pain during hamstring stretching in healthy young subjects. Thirty-one participants (n=31) were randomly divided into 2 groups: an experimental group (EG, n=15), which was submitted to hamstring stretching exercise for 180 sec, and the control group (n=16), which was not exercised. Skin temperature (TsK) was collected in subjects' posterior thigh using a thermographic camera at 7 time points with intervals of 30 sec each: T0 (rest), T30, T60, T90, T120, T150, and T180. In addition, the knee range of motion, passive stiffness and muscle soreness (visual analogue scale) were recorded during the stretching procedure at the same times. The EG presented a small but progressive increase in TsK (30.8°C±0.6°C to 31.1°C±0.6°C, P<0.01), with an increased range of motion (P<0.01) and muscle soreness levels (P<0.01) during the gradual hamstring stretching protocol. Additionally, there was an increment of passive stiffness (P<0.01) that was partially reversed toward the end of the stretching period. This research presents evidence that constant, passive and progressive stretching of the hamstrings progressively increased the local TsK by about 0.3°C after 180 sec of stretching. These findings may help to understand the effects of repeated stress on the muscle-tendon unit and the role of muscle stretching in warm-up protocols and rehabilitation.