The effects of two key experimental parameters on the measured nanomechanical properties of lamellar and interlamellar tissue were examined in dehydrated rabbit cancellous bone. An anhydrous sample preparation protocol was developed to maintain surface integrity and produce RMS surface roughnesses ∼10 nm (5 × 5-μm2 area). The effects of surface roughness and maximum nanoindentation load on the measured mechanical properties were examined in two samples of differing surface roughness using maximum loads ranging from 250 to 3000 μN. As the ratio of indentation depth to surface roughness decreased below ∼3:​1, the variability in material properties increased substantially. At low loads, the indentation modulus of the lamellar bone was ∼20% greater than that of the interlamellar bone, while at high loads the measured properties of both layers converged to an intermediate value. Relatively shallow indentations made on smooth surfaces revealed significant differences in the properties of lamellar and interlamellar bone that support microstructural observations that lamellar bone is more mineralized than interlamellar bone.