The relationship between electrical and morphological properties of annealed Nafion samples is investigated by X-ray diffraction (XRD), small angle X-ray scattering (SAXS), atomic force microscopy (AFM), and impedance spectroscopy. Experimental data reveal that the heat treatment at high temperature (T similar to 130-140 degrees C) with low relative humidity (RH similar to 0%) results in significant changes of Nafion such as increased crystallinity and decreased average distance of hydrophilic domains. Such effects were practically absent when the same heat treatment was carried out at high RH similar to 100%. The effects of annealing with controlled RH were reflected in the polymer electrolyte fuel cell (PEFC) tests in which the measured performance was markedly reduced for Nafion samples annealed at low RH. Such a feature was related to decreased microstructural stability, water sorption and proton conductivity of the annealed membrane. The observed effects are relevant to evaluate degradation of Nafion during both fuel cell assembly and harsh PEFC operating conditions. Moreover, the experimental results contribute to advance the understanding of Nafion's properties at high temperature for the development of high-performance ionomer membranes. (C) 2016 Elsevier Ltd. All rights reserved.