Background To properly study knee kinetics, kinematics and the effects of injury and surgical treatment in vitro, the knee should be constrained as little as possible, while imposing physiological loads. A novel dynamic biomechanical knee system (BKS) is presented here. The aim of this study was to test the feasibility and reproducibility of the system and demonstrate its features with an Anterior Cruciate Ligament (ACL) lesion model. Methods Six goat knees were used in the current study. Flexion and extension simulating gait was imposed by a servo-motor, while normal joint load was applied by two artificial muscles. Intra-class correlation coefficients (ICCs) were assessed for inter-test measures, while paired t-tests were performed for comparison between intact knees and knees with ACL-lesion. Results The ICC's for inter-test measures based on all six goat knees were excellent: varus/valgus: ICC = 0.93; rotation: ICC = 0.94 (all p < 0.01), and translation in frontal (x)-, side (y)- and upward (z)-direction (ICC = 0.90, 0.88 & 0.94) (all p < 0.01). A significant increase in joint center movement was found in knees after creating an ACL-lesion (p = 0.018): translation increased more than two-fold in frontal (p = 0.016), side (p = 0.004) and upward (p = 0.018) direction. Conclusions Five degrees of motion were reproducibly assessed in the intact joint, suggesting that the goat knee may find its natural pathway when loaded in the BKS. The novel five-degrees-of-freedom knee system allows a detailed study of the effect of a diversity of defects and surgical treatments on knee biomechanics under physiological loading conditions.