This study examined whether a knee flexor isometric contraction at 20% of maximal voluntary isometric contraction until exhaustion would alter the biceps femoris long head (BFlh) and semitendinosus (ST) active stiffness, assessed using ultrasound-based shear wave elastography. Twelve healthy individuals participated in 2 sessions separated by 7 days. Time to exhaustion was similar in both sessions (day 1: 443.8 ± 192.5 s; day 2: 474.6 ± 131.7 s; p = 0.323). At the start of the fatigue task, the ST showed greater active stiffness than the BFlh (p < 0.001), with no differences between days (p = 0.08). The ST active stiffness then decreased from 40% of the task time to exhaustion (- 2.2 to - 13.3%, p = 0.027) until the end of the task (- 16.1 to - 22.9%, p = 0.012), while no significant changes were noted in the BFlh (p = 0.771). Immediately after the fatigue task, a decrease in active stiffness was observed in the ST (- 11.8 to - 17.8%, p < 0.001), but not in the BFlh (p = 0.551). Results were consistent between the 2 testing sessions (p = 0.07-0.959). The present results indicate that fatigue alters the hamstring active stiffness pattern.