The effects of static stretching are influenced by prescribed and applied loads of stretching. The prescribed load is calculated from the stretching duration and intensity, whereas the applied load is assessed from the force of static stretching exerted on the targeted muscle. No previous study has investigated the prescribed and applied loads of static stretching on the muscle-tendon unit stiffness simultaneously. Therefore, the purpose of the present study was to examine the acute effects of the prescribed and applied load of static stretching on the change in the muscle-tendon unit stiffness of the hamstrings by using different intensities and durations of static stretching. Twenty-three participants underwent static stretching at the intensity of high (50 seconds, 3 sets), moderate (60 seconds, 3 sets), and low (75 seconds, 3 sets), in random order. The parameters were the range of motion, passive torque, and muscle-tendon unit stiffness. These parameters were measured before stretching, between sets, and immediately after stretching by using a dynamometer machine. The static stretching load was calculated from the passive torque during static stretching. The muscle-tendon unit stiffness decreased in high- and moderate-intensity after 50 (p < 0.01, d = -0.73) and 180 seconds (p < 0.01, d = -1.10) of stretching respectively, but there was no change in low-intensity stretching for 225 seconds (p = 0.48, d = -0.18). There were significant correlations between the static stretching load and relative change in the muscle-tendon unit stiffness in moderate- (r = -0.64, p < 0.01) and low-intensity (r = -0.54, p < 0.01), but not in high-intensity (r = -0.16, p = 0.18). High-intensity static stretching was effective for a decrease in the muscle-tendon unit stiffness even when the prescribed load of static stretching was unified. The applied load of static stretching was an important factor in decreasing the muscle-tendon unit stiffness in low- and moderate-intensity static stretching, but not in high-intensity stretching.