A direct methanol fuel cell (DMFC) needs to be activated to achieve its maximum performance. The activation procedure includes a pre-treatment and an in situ activation procedure. Along the activation procedure the membrane electrode assembly (MEA) experiments significant changes that are studied by electrochemical impedance spectroscopy (EIS), polarization curves and methanol crossover measurements. It is shown that the activation procedure makes the proton conductivity of the PEM to increase as well as the catalyst area and activity. The DMFC power density increases from 8.8 mWcm(-2) to 22.4mWcm(-2) at 55 degrees C along the activation procedure. The Design of Experiments (DoE) methodology was then applied to optimize the in situ activation, where loading cycles were employed. The factors considered for the experimental design were the temperature, the loading and the cathode air pressure. The maximum power density response was optimized using a central composite design (CCD). It was also verified that the potential was the most significant factor. Finally, the in situ loading cycles procedure was critically compared with other in situ activation procedures reported in the literature. It was concluded that the hydrogen conditioning and the in situ loading cycles procedure led to the best performance of the DMFC. (C) 2011 Elsevier Ltd. All rights reserved.