The influence of various experimental conditions on the elastic modulus and hardness of human intertubular dentin was studied using instrumented nanoindentation at room temperature. The conditions studied were: dry (chemically dehydrated) versus wet (prepared and nanoindented under Hank's balanced salt solution (HBSS)), the influence of long-term storage under HBSS at ca. 4°C and the influence of deep-freezing under dry and wet conditions. The reversibility of chemical dehydration and the consequences of multiple deep-freezing cycles were also investigated. Two premolars of a 12-year-old human male were chosen for evaluation. The absolute values of the mechanical properties differed by a factor of ∼2 and ∼3 between dry and wet states, whereas trends were conserved. Deep-freezing wet dentin samples weakened their mechanical properties by 20–28%, whereas dehydrated samples suffered no damage. This may be due to mechanical damage via the volume increase in water freezing inside the tubules.