Background: Limited evidence exists on neuromuscular risk factors for anterior cruciate ligament injuries, with most work mainly focussing on the strength of hamstrings and quadriceps muscles. In this prospective pilot study, we explored if neuromuscular activation patterns of the quadriceps and hamstrings during a drop vertical jump influence anterior cruciate ligament injury risk.Methods: Forty-six female athletes performed a drop vertical jump at baseline. Injuries were monitored throughout a one year follow-up. Neuromuscular activation patterns of the vastus medialis, vastus lateralis, hamstrings medialis and hamstrings lateralis, and selected landing kinematic and kinetic profiles (knee flexion, knee abduction and hip flexion angles, and knee abduction moments), were compared between athletes who sustained a non-contact anterior cruciate ligament injury and those who remained injury-free. EMG vector fields were created to represent neuromuscular activation patterns of muscle pairs around the knee joint rather than only considering individual muscle activations, and compared using Statistical Parametric Mapping.Results: Four athletes sustained an anterior cruciate ligament injury. Significantly greater {hamstrings medialis, hamstrings lateralis}, {vastus lateralis, hamstrings lateralis} and {hamstrings lateralis, vastus medialis} activations, mainly due to greater hamstrings lateralis activation, were found in the injured group around peak loading and just before take off (P < 0.001). No group differences were found in knee flexion, knee abduction and hip flexion angles, or knee abduction moments.Conclusions: This pilot study revealed initial evidence that athletes already showed altered neuromuscular activation patterns prior to sustaining an anterior cruciate ligament injury, namely increased lateral and posterior muscle activations.