Purpose Valgus deformity presents a particular challenge in total knee arthroplasty. This condition regularly leads to contractures of the lateral capsular ligament complex and to overstretching of the medial ligamentous complex. Reconstruction of the knee joint kinematics and anatomy often requires lateral release. However, data on how such release weakens the stability of the knee are missing in the literature. This study investigated the effects of sequential lateral release on the collateral stability of the ligament complex of the knee in vitro. Methods Ten knee prostheses were implanted in 10 healthy cadaveric knee joints using a navigation device. Soft tissue lateral release consisted of five release steps, and stiffness and stability were determined at 0, 30, 60 and 90 degrees flexion after each step. Results Soft tissue lateral release increasingly weakened the ligament complex of the lateral compartment. Because of the large muscular parts, the release of the iliotibial band and the M. popliteus had little effect on the stability of the lateral and medial compartment, but release of the lateral ligament significantly decreased the stability in the lateral compartment over the entire range of motion. Stability in the medial compartment was hardly affected. Conversely, further release of the posterolateral capsule and the posterior cruciate ligament led to the loss of stability in the lateral compartment only in deep flexion, whereas stability decreased significantly in the medial compartment. Conclusion Our study shows for the first time the association between sequential lateral release and stability of the ligamentous complex of the knee. To maintain the stability, knee surgeons should avoid releasing the entire lateral collateral ligament, which would significantly decrease stability in the lateral compartment.