Biomechanical tests of human femora have shown that small variations of the loading direction result in significant changes in measured bone mechanical properties. However, the heterogeneity in geometrical and bone tissue properties does not make human bones well suited to reproducibly assess the effects of loading direction on stiffness and strength. To precisely quantify the influence of loading direction on stiffness and strength of femora loaded at the femoral head, we tested femora from C57BIJ6 inbred mice. We developed an image-based alignment protocol and investigated the loading direction influence on proximal femur stiffness and strength. An aluminum femoral phantom and C57BIJ6 femora were tested under compression with different loading directions. Both tests, with the aluminum phantom and the murine bones, showed and quantified the linear dependence of stiffness on loading direction: a 5' change in loading direction resulted in almost 30% change in stiff ness. Murine bone testing also revealed and quantified the variation in strength due to loading direction: 5' change in loading direction resulted in 8.5% change in strength. In conclusion, this study quantified, for the first time, the influence of misalignment on bone stiffness and strength for femoral head loading. We showed the extreme sensitivity of this site regarding loading direction.