Abstract This paper presents the diagnostic results of single polymer electrolyte membrane fuel cell assemblies characterized by polarization curves. Single PEM fuel cell assemblies were investigated through accelerated voltage cycling test at different values of relative humidity. The fuel cells are tested at different humidity level. The cells are discussed in this paper with analysis results at different relative humidity at atmospheric pressure. This represents a nearly fully humidified, a moderately humidified, and a low humidified condition, respectively. This technique is useful for diagnosing the main sources of loss in MEA development work, especially for high temperature/low relative humidity operation where several sources of loss are present simultaneously. All the fuel cells showed better performance in terms of limiting current density value through polarization curves when oxygen was fed to the cathode side of each cell instead of air. The results indicate that the performance of the fuel cell could be depressed significantly by decreasing RH from 100 to 33%. Decrease in RH can result in slower electrode kinetics, including electrode reaction and mass diffusion rates, and higher membrane resistance.