Understanding the pathophysiology of metabolic bone disease requires the characterization of both the quantity as well as the quality (i.e., microarchitecture and material properties) of the bone tissue. Nanoindentation provides a powerful yet simple method to measure the nano/micro mechanical properties of bone, but no uniform testing methodology exists. This study examines the effects of embedding materials, rate and depth of indentation, and storage time on the measured modulus. Nineteen trabecular bone samples were evaluated for the study. Although there was an 8-fold increase in the stiffness of the soft to hard epoxy, bone tissue modulus was not affected by the stiffness of the embedding materials, but hardness was affected by both the embedding material modulus, for example from 0.70 ± 0.20 GPa (ME_low) to 0.45 ± 0.21 GPa (ME_Med) (p < 0.01), and viscosity (p < 0.01). No significant differences were found with regard to the tested rates and depths of indentation for either elastic modulus or hardness. The tissue modulus tested at the 6-month time point was significantly greater in comparison with that tested at 0 or 3 months (p < 0.01). The hardness, however, did not significantly change over the span of 6 months. The results show that while nanoindentation is powerful, it is particularly sensitive to certain testing variables.