Trabecular bone tissue is a three-dimensional structure that is difficult to duplicate with in vitro cell cultures or animal models. In an attempt to better understand the underlying mechanisms of tissue response to load, a system to load isolated bone preparations was developed. This ex vivo bone culture and loading system, given the name of ZETOS, compressively loads trabecular bone ( diameter, height) to evaluate its morphological and physiological responses while keeping cells viable. Compliance of the system may change with time, thus requiring recalibration. The purpose of this research was to develop and validate a recalibration protocol for the ZETOS system. Ten reference bodies (RBs) were designed and machined out of aluminum 7075-T6, with a structural rigidity range representative of trabecular bone (, or apparent elastic modulus of ). Finite element analysis (FEA) was used to calculate the rigidity of each RB and was validated with physical testing in a universal testing machine. Results from FEA were then used to calibrate the system and relate force, piezoelectric actuator expansion, and specimen compressive deformation through a surface generated by spline interpolation, thus creating a calibration table. Calibration of ZETOS was verified by testing the RBs as well as three custom-made, metal springs and comparing measured rigidity to that calculated by FEA. Mean percent difference of FEA results with respect to those from physical testing was 3.28%. The mean percent difference of RB rigidity found with ZETOS with respect to rigidity found with FEA was 1.12% and for the metal springs, the mean percent difference was 1.74%. The calibration procedure for the ZETOS bone loading system has been successfully applied and verified. The use of RBs and FEA allows users to easily and periodically evaluate and recalibrate the system. Accuracy in studies of human bone mechanotransduction in a controlled environment can therefore be achieved. The recalibration procedure is relevant for other ZETOS users and may serve as the basis for calibration of other testing systems for small specimens of compliant materials.