Inability to accurately and objectively assess the mechanical properties of healing fractures in vivo hampers clinical fracture management and research. We describe a method to monitor fracture stiffness during healing in a clinical research setting by detecting changes in fracture displacement using radiostereometric analysis and simultaneously measuring applied axial loads. A method was developed for load application, positioning of the patient, and radiographic setup to establish the technique of differentially loaded radiostereometric analysis (DLRSA). A DLRSA examination consists of radiostereometric analysis radiographs taken without load (preload), under different increments of load, and without load (postload). Six patients with distal femur fractures had DLRSA examinations at 6, 12, 18, and 26 weeks postoperatively. The DLRSA method was feasible in a clinical setting. The method provides objective and quantifiable data for internally fixed fractures and may be used in clinical research as a tool to monitor the in vivo stiffness of healing femoral fractures managed with nonrigid internal fixation.