The purpose of this investigation was to design and verify the capabilities of an in vitro loading-measurement system that mimics in vivo unconstrained three dimensional (3D) relative motion between long bone ends, applies uniform load components over the entire length of a test specimen, and measures 3D relative motion between test segment ends to directly determine test segment construct stiffness free of errors due to potting-fixture-test machine finite stiffness.Intact equine cadaveric radius bones, which were subsequently osteotomized/ostectomized and instrumented with bone plates were subjected to non-destructive axial, torsion, and 4-point bending loads through fixtures designed to allow unconstrained components of non-load associated 3D relative motion between radius ends. 3D relative motion between ends of a 50 mm long test segment was measured by an infrared optical tracking system to directly determine its stiffness. Each specimen was then loaded to ultimate failure in either torsion or bending. Cortical bone cross-section diameters and published bone biomechanical properties were substituted into classical mechanics equations to predict the intact test segment theoretical stiffness for comparison and thus loading-measurement system verification.Intact measured stiffness values were the same order of magnitude as theoretically predicted. The primary component of relative motion between ends of the test segment corresponded to that of the applied load with the other 3D components being evident and consistent in relative magnitude and direction for unconstrained loading of an unsymmetrical double plate oblique fracture configuration. Bone failure configurations were reproducible and consistent with theoretically predicted.The 3D loading-measurement system designed: a) mimics unconstrained relative 3D motion between radius ends that occurs in clinical situations, b) applies uniform compression, torsion, and 4-point bending loads over the entire length of the test specimen, c) measures interfragmentary 3D relative motion between test segment ends to directly determine stiffness thus being void of potting-fixture-test machine stiffness error, and d) has the resolution to detect differences in the 3D motion and stiffness of intact as well osteotomized-instrumented and ostectomized-instrumented equine radii.