Cross-sectional area, stiffness, viscoelastic stress relaxation, stretch tolerance and EMG activity of the human hamstring muscle group were examined in endurance-trained athletes with varying flexibility. Subjects were defined as tight (n = 10) or normal (n = 8) based on a clinical toe-touch test. Cross-sectional area was computed from magnetic resonance imagining (MRI) images. Torque (Nm) offered by the hamstring muscle group, electromyographic (EMG) activity, knee joint angle and velocity were continuously monitored during two standardized stretch protocols. Protocol 1 consisted of a slow stretch at 0.087 rad/s (dynamic phase) to a pre-determined final angle followed by a 90-s static phase. In the dynamic phase final angle and stiffness was lower in tight (28.0+/-2.9 Nm/rad) than normal subjects (54.9+/-6.5 Nm/rad), P<0.01. In the static phase tight subjects had lower peak (15.4+/-1.8 Nm) and final torque (10.8+/-1.6 Nm) than normal subjects (31.6+/-4.1 Nm, 24.1+/-3.7 Nm, respectively)(P<0.01), but torque decline was similar. Protocol 2 consisted of a slow stretch to the point of pain and here tight subjects reached a lower maximal angle, torque, stiffness and energy than normal subjects (P<0.01). On the other hand, stiffness was greater in tight subjects in the common range (P<0.01). Cross-sectional area of the hamstring muscles and EMG activity during the stretch did not differ between the groups. However, lateral hamstring cross-sectional area was positively related to mid-range stiffness (P<0.05), but inversely related to final stiffness, peak torque and the toe-touch test (P<0.01). Final angle and peak torque in protocol 1 combined to improve the predictability of the toe-touch test (R2=0.77, P<0.001). These data show that the toe-touch test is largely a measure of hamstring flexibility. Further, subjects with a restricted joint range of movement on a clinical toe-touch test have stiffer hamstring muscles and a lower stretch tolerance.