To examine musculotendinous stiffness (MTS) and ankle joint range of motion (ROM) in men and women following an acute bout of passive stretching. Eight men (mean ± SD age = 20.3 ± 2.0 yrs; body mass = 78.8 ± 12.3 kg; height = 176.6 ± 5.8 cm) and nine women (age = 21.0 ± 2.4 yrs; body mass = 61.7 ± 7.4 kg; height = 166.3 ± 7.5 cm) volunteered for this study. To avoid any menstrual cycle differences, the women were all tested during menses. Each subjects foot was stabilized in a custom-built apparatus designed to measure plantar flexion force with a knee joint angle of 180° and ankle joint angle of 90°. The apparatus was also attached to a powered isokinetic dynamometer that stretched the plantar flexor muscles by passively dorsiflexing the foot at 5°·s−1 until a constant-torque threshold was achieved and held at a point of discomfort as acknowledged by the subject. Nine repetitions of each stretch were held for 135 s with 10 s of rest between repetitions. Before and after the stretching, a maximal, passive flexibility assessment was performed, in which subjects were asked to relax while their foot was maximally dorsiflexed at 5°·s−1. Passive torque and position were recorded from the isokinetic dynamometer. To calculate MTS, the ankle joint angle (°) and torque (Nm) signals were sampled at 1 KHz during the flexibility assessments and plotted as torque-angle curves (i.e., stress-strain curves). Each subject's curve was fit with a 4th-order polynomial regression model (Nordez et al. 2006, Clin Biomech, 21:755-760), and MTS was calculated as the slope (Nm°-1) of the tangent to the curve at a 90° joint angle. MTS decreased by 15.4% (p < 0.001) in men and 36.7% (p < 0.001) in women from pre- to post-stretching. Passive range of motion (PROM) increased by 3.1% (p = 0.018) in men and 7.3% (p = 0.001) in women from pre- to post-stretching. No differences (p > 0.05) were observed between men and women for PROM and MTS prior to and post-stretching. However, the percent change scores from pre- to post-stretching for MTS (p = 0.021) and PROM (p = 0.036) were greater for the women than the men. These findings suggested that 20 min of constant-torque passive stretching increased the PROM and decreased the MTS of the plantar flexors in both men and women, although the magnitude of the change was greater for the women. The greater decrease in MTS and increase in PROM for women may have been due to gender-specific differences in viscoelastic creep during the constant-torque stretching procedures. It has been suggested that a decrease in MTS reduces the total amount of strain through a given ROM, which may reduce the risk of strain injuries. Therefore, the findings of this study suggest that men may have to stretch for a longer duration or at a greater intensity to achieve similar increases in ROM and decrease in MTS as women.