Dynamic stretching (DS) is often performed during warm-up to help avoid hamstring muscle injuries, increase joint flexibility, and optimize performance. We examined the effects of DS of the hamstring muscles on passive knee extension range of motion (ROM), passive torque (PT) at the onset of pain (as a measure of stretch tolerance), and passive stiffness of the muscle-tendon unit over an extended period after stretching. Twenty-four healthy subjects participated, with 12 each in the experimental and control groups. Stretching was performed, and measurements were recorded using an isokinetic dynamometer pre-intervention, and at 0, 15, 30, 45, 60, 75, and 90 min post-intervention. DS consisted of ten 30-s sets of 15 repetitions of extension and relaxation of the hamstrings. ROM increased significantly (range, 7%–10%) immediately after DS, and the increase was sustained over 90 min. PT at the onset of pain also increased immediately by 10% but returned to baseline by 30 min. Passive stiffness decreased significantly (range, 7.9%–16.7%) immediately after DS, and the decrease was sustained over 90 min. Post-DS values were normalized to pre-DS values for the respective outcomes in both groups. ROM was significantly higher (range, 7.4%–10%) and passive stiffness was significantly lower (range, 5.4%–14.9%) in the experimental group relative to the control group at all time points. Normalized PT values at the onset of pain were significantly higher in the experimental group at 0–15 min than in the controls, but the differences were smaller at 30–45 min and not significant thereafter. We conclude that DS increases ROM and decreases passive stiffness in a sustained manner, and increases PT at the onset of pain for a shorter period. Overall, our results indicate that when performed prior to exercise, DS is beneficial for the hamstring muscles in terms of increasing flexibility and reducing stiffness.

• Effect of dynamic stretching (DS) on hamstring muscle flexibility was examined.

• DS increased range of motion and stretch tolerance, and decreased passive stiffness.

• Stretch tolerance returned more rapidly after DS than other measurement indexes.

• DS performed prior to exercise is useful for improving hamstring muscle flexibility.