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Standard clinical MRI techniques provide morphologic insights into knee joint pathologies, yet do not allow evaluation of ligament functionality or joint instability. We aimed to study valgus stress MRI, combined with sophisticated image post-processing, in a graded model of medial knee joint injury. To this end, eleven human cadaveric knee joint specimens were subjected to sequential injuries to the superficial medial collateral ligament (sMCL) and the anterior cruciate ligament (ACL). Specimens were imaged in 30° of flexion in the unloaded and loaded configurations (15 kp) and in the intact, partially sMCL-deficient, completely sMCL-deficient, and sMCL- and ACL-deficient conditions using morphologic sequences and a dedicated pressure-controlled loading device. Based on manual segmentations, sophisticated 3D joint models were generated to compute subchondral cortical distances for each condition and configuration. Statistical analysis included appropriate parametric tests. The medial compartment opened gradually as a function of loading and injury, especially anteriorly. Corresponding manual reference measurements by two readers confirmed these findings. Once validated in clinical trials, valgus stress MRI may comprehensively quantify medial compartment opening as a functional imaging surrogate of medial knee joint instability and qualify as an adjunct diagnostic tool in the differential diagnosis, therapeutic decision-making, and monitoring of treatment outcomes.