Anterior cruciate ligament (ACL) rupture leads to mechanical and functional knee instability. Functional instability is likely attributable to a sensorimotor deficit. In previous studies, a method has been introduced which allows this deficit to be objectively assessed using ACL-hamstring reflex measurements. There is evidence that subjectively stable and unstable patients with ACL rupture can be objectively distinguished by this method. This study on 34 men was conducted to investigate the reliability of this method on the basis of several factors (intra-individual reproducibility, side-to-side differences, inter-examiner reliability, audiovisual stimuli, fatigue, height, weight, physical fitness, and level of activity at work). The role of gender was assessed in 20 men and 20 women. None of the factors had a relevant influence on reflex responses. The tests were reproducible and reliable. The method described here can thus be used for further research and the clinical diagnosis of functional instability following ACL rupture.