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We propose using the single-leg squat-and-hold (SLSH) task with kinematic analysis to objectively measure dynamic knee stability after anterior cruciate ligament (ACL) injury. There are three objectives of this study: to compare the knee kinematics of ACL-deficient patients and healthy controls by capturing knee wobbling during the SLSH task, to detect kinematic changes after ACL reconstruction, and to correlate the kinematic variables with self-reported knee function. Twenty-five ACL-deficient participants and 18 healthy matched participants were recruited. The knee kinematics involving both the magnitudes and frequency of motion fluctuation was captured during SLSH by 3D motion analysis system (Vicon). Compared to the limbs of the control participants, the ACL involved limbs exhibited a greater range of flexion-extension (4.33 ± 1.96 vs. 2.73 ± 1.15; p = 0.005) and varus-valgus (2.52 ± 0.99 vs. 1.36 ± 0.42; p < 0.001). It also inhibited higher frequency of flexion-extension (4.87 ± 2.55 vs. 2.68 ± 1.23; p = 0.003) and varus-valgus (3.83 ± 2.59 vs. 1.42 ± 0.55; p < 0.001). The range of flexion-extension (4.50 ± 2.24 vs. 2.90 ± 1.01; p = 0.018), frequency of flexion-extension (4.58 ± 2.53 vs. 3.05 ± 1.80; p = 0.038) and varus-valgus (3.46 ± 2.11 vs. 1.80 ± 1.23; p = 0.022) was reduced after ACL reconstruction. Increased frequency of knee varus-valgus was correlated with lower IKDC score (r = −0.328; p = 0.034). Knee wobbling was more prominent in ACL-deficient patients, which was associated with poor knee function. SLSH task with kinematic analysis appears to be a potential assessment method for monitoring dynamic knee stability after ACL injury. [ABSTRACT FROM AUTHOR]