The determination of bone mechanical properties remains crucial, especially to feed up numerical models. An original methodology of inverse analysis has been developed to determine the longitudinal elastic modulus of femoral cortical bone. The method is based on a numerical twin of a specific three-point bending test. It has been designed to be reproducible on each test result. In addition, the biofidelity of the geometric acquisition method has been quantified. As the assessment is performed at the scale of a bone shaft segment, the Young's modulus values obtained (between 9518.29 MPa and 14181.15 MPa) are considered average values for the whole tissue, highlighting some intersubject variability. The material microstructure has also been studied through histological analysis, and bone-to-bone comparisons highlighted discrepancies in quadrants microstructures. Furthermore, significant intrasubject variability exists since differences between the bone's medial-lateral and anterior–posterior quadrants have been observed. Thus, the study of microstructures can largely explain the differences between the elastic modulus values obtained. However, a more in-depth study of bone mineral density would also be necessary and would provide some additional information. This study is currently being setup, alongside an investigation of the local variations of the elastic modulus.