Objective To investigate the effect of sustained fatigue on the passive and active biomechanical characteristics of the knee. Methods Twenty-seven healthy university students were recruited to perform A fatigue experiment using an isokinetic dynamometer. The fatigue experiment included three fatigue cycles with more than 30 sustained quadriceps submaximal voluntary isometric contractions per cycle. The maximum passive torque, mean maximum voluntary contraction (MVC) torque, integrated electromyography of the vastus lateralis and biceps femoris, and co-contraction index (CI) at different fatigue cycles were compared. Results The maximum passive torque decreased significantly only after the 3rd fatigue cycle (P<0.05). The mean MVC torque and integrated electromyography of the vastus lateralis and biceps femoris decreased significantly during the 1st, 2nd, and 3rd fatigue cycles (P<0.05). However, the CI did not change significantly (P>0.05). Conclusions Sustained fatigue intervention with 90-fold isometric contraction training of The quadriceps significantly affected the active and passive biomechanical properties of the knee. After fatigue intervention, the resistance of the knee joint to passive flexion and extension decreased. With fatigue intervention, the active contraction ability of the quadriceps decreased, and the activation of the quadriceps femoris and hamstring muscles also decreased; however, the co-contraction level of the two muscle groups remained unchanged. This mild muscle group co-contraction pattern is beneficial for maintaining a benign mechanical loading environment in the knee joints. These results help us understand the active and passive biomechanical properties of knee joints after fatigue.