Purpose Balancing mobile-bearing (MB) unicondylar knee arthroplasty (UKA) is challenging. If performed improperly, potential complications include pain, implant loosening, or progression of osteoarthritis in the preserved compartment. The purpose of this study was to document effects of improper balancing on knee kinematics and joint contact stress. It was hypothesized that over-stuffing would lead to more valgus and higher lateral contact force. Methods Seven fresh-frozen cadaver legs were mounted in a kinematic rig that applied three motion patterns to the specimens: passive flexion–extension, open chain extension, and squatting. During testing, an infrared camera system recorded the trajectories of markers rigidly attached to femur and tibia, while a pressure sensor measured contact pressure in the lateral compartment. Prior computer tomography scans allowed identification of coordinate frames of the bones and calculations of anatomical rotations and translations. Collateral ligament strains were calculated, and quadriceps forces recorded. Following testing on the native knee, a medial MB UKA was implanted in each specimen and all motion trials were repeated. Three inlay thicknesses were tested to simulate optimal balancing as well as under- (1 mm thinner) and over-stuffing (1 mm thicker) of the medial compartment relative to the optimal thickness. Results Under-stuffing of the medial compartment leads to kinematics closest to the native knee. Subjectively balanced and over-stuffed MB UKA knees were in more valgus. Lateral peak contact stress was higher from mid- to deep flexion following UKA in all three tested states; however, these results were not significant. Peak strain in the superficial medial collateral ligament (sMCL) was significantly higher in MB UKA, regardless of the inlay thickness mainly in mid-flexion. Inlay thickness had no significant impact on measured quadriceps force during squatting. Conclusion The results underline the importance of optimal balancing. Over-stuffing should be avoided as it results in the largest kinematic changes relative to the native condition and induces higher strains in the sMCL. Based on the kinematic findings, it is advisable to use thinner inlays, as long as this is not compromising stability or risking inlay luxation.