This work reports results from quasi-static nanoindentation measurements of iron, in the un-strained state and subjected to 15% tensile pre-straining at room temperature, 125 °C and 300 °C, in order to extract room temperature hardness and elastic modulus as a function of indentation depth. The material is found to exhibit increased disposition for pile-up formation due to the prestraining, affecting the evaluation of the mechanical properties of the material. Nanoindentation data obtained with and without pre-straining are compared with bulk tensile properties derived from the tensile pre-straining tests at various temperatures. A significant mismatch between the hardness of the material and the tensile test results is observed and attributed to increased pile-up behaviour of the material after pre-straining, as evidenced by atomic force microscopy. The observations can be quantitatively reconciled by an elastic modulus correction applied to the nanoindentation data, and the remaining discrepancies explained by taking into account that strain hardening behaviour and nano-hardness results are closely affected by dynamic strain ageing caused by carbon interstitial impurities, which is clearly manifested at the intermediate temperature of 125 °C.