Static stretching is widely applied in various disciplines. However, the acute effects of different durations of stretching are unclear. Therefore, this study was designed to investigate the acute effects of different stretching durations on muscle function and flexibility, and provide an insight into the optimal duration of static stretching. This randomized crossover trial included 24 healthy students (17 men and 7 women) who stretched their right hamstrings for durations of 20, 60, 180, and 300 seconds in a random order. The following outcomes were assessed using an isokinetic dynamometer as markers of lower-limb function and flexibility: static passive torque (SPT), dynamic passive torque (DPT), stiffness, straight leg raise (SLR), and isometric muscle force. Static passive torque was significantly decreased after all stretching durations (p < 0.05). Static passive torque was significantly lower after 60, 180, and 300 seconds of stretching compared with that after 20-second stretching, and stiffness decreased significantly after 180- and 300-second stretching (p < 0.05). In addition, DPT and stiffness were significantly lower after 300 seconds than after 20-second stretching (p < 0.05), and SLR increased significantly after all stretching durations (p < 0.05). Straight leg raise was higher after 180- and 300-second stretching than after 20-second stretching and higher after 300-second stretching than after 60-second stretching (p < 0.05). Isometric muscle force significantly decreased after all stretching durations (p < 0.05). Therefore, increased duration of stretching is associated with a decrease in SPT but an increase in SLR. Over 180 seconds of stretching was required to decrease DPT and stiffness, but isometric muscle force decreased regardless of the stretching duration. In conclusion, these results indicate that longer durations of stretching are needed to provide better flexibility.