The current study aimed using a two-experiment musculoskeletal simulation-based approach, measuring anterior cruciate ligament (ACL) biomechanics, knee joint kinematics, and lower extremity joint loading to examine the effects of both a prophylactic knee sleeve on (1) a sport-specific change of direction movement in female footballers and (2) a single leg landing in male footballers. Experiment 1 examined 12 female university first team level footballers (age [Formula: see text] years, height [Formula: see text][Formula: see text]m, body mass [Formula: see text][Formula: see text]kg) undertaking a [Formula: see text] cutting movement in sleeve and no-sleeve conditions. Experiment 2 examined 10 male university first team level footballers (age [Formula: see text] years, height [Formula: see text][Formula: see text]m, body mass [Formula: see text][Formula: see text]kg) undertaking a single leg drop jump landing in sleeve and no-sleeve conditions. In each experiment, data was collected in a biomechanics laboratory and three-dimensional (3D) motion capture and ground reaction force (GRF) information was collected. 3D kinematics, 3D knee kinetics, and ACL ligament forces/strains were measured using musculoskeletal simulation, and participants were also asked to subjectively rate the knee sleeve in terms of both comfort and stability. Experiment 1 showed that the sleeve condition was associated with greater ACL strain ([Formula: see text]% and no-[Formula: see text]%) and forces ([Formula: see text] BW and no-[Formula: see text] BW). In addition, the brace condition also enhanced lateral compressive tibiofemoral ([Formula: see text] BW and no-[Formula: see text] BW) and total compressive tibiofemoral force ([Formula: see text] BW and no-[Formula: see text] BW). Finally, for the subjective ratings, participants indicated that the knee sleeve significantly improved perceived comfort and stability. Experiment 2 did not reveal any statistical differences between knee sleeve and no-sleeve conditions nor any effects of the knee sleeve on subjective ratings of comfort or stability. Therefore, the findings from the current investigation suggest that the prophylactic knee sleeve examined in the current investigation does not appear to reduce the biomechanical parameters linked to the aetiology of knee pathologies in male/female footballers.