Measuring the microscopic mechanical properties of bone tissue is important in support of understanding the etiology and pathogenesis of many bone diseases. Knowledge about these properties provides a context for estimating the local mechanical environment of bone related cells that coordinate the adaptation to loads experienced at the whole organ level. The objective of this study was to determine the effects of experimental testing parameters on nanoindentation measures of lamellar-level bone mechanical properties. Specifically, we examined the effect of specimen preparation condition, indentation depth, repetitive loading, time delay, and displacement rate. The nanoindentation experiments produced measures of lamellar elastic moduli for human cortical bone (average value of for osteons and for interstitial bone tissue). In addition, the hardness measurements produced results consistent with data in the literature (average for osteons and for interstitial bone tissue). Consistent modulus values can be obtained from a 500-nm-deep indent. The results also indicated that the moduli and hardnesses of the dry specimens are significantly greater (22.6% and 56.9%, respectively) than those of the wet and wet and embedded specimens. The latter two groups were not different. The moduli obtained at a loading rate were significantly lower than the values at the 10- and loading rates while the 10- and rates were not significantly different. The hardness measurements showed similar rate-dependent results. The preliminary results indicated that interstitial bone tissue has significantly higher modulus and hardness than osteonal bone tissue. In addition, a significant correlation between hardness and elastic modulus was observed.