The organic matter consumption and hydrogen production rate were evaluated in a two-chamber microbial electrolysis cell (MEC). Three chemical oxygen demand (COD) concentration levels (400, 600 and 1200 mg/L) were tested. The COD was composed of a mixture of volatile fatty acids (VFAs) present in the effluent of a dark fermentation process. The two levels of voltage studied were 350 and 550 mV. The performance of the MEC was evaluated using either an anionic (AEM) or cationic exchange membrane (CEM). The robustness of the MEC was tested using two dark fermentation effluents, one with VFAs and another containing 1100 mg/L glucose. The highest production rates (81 mL/L/day) were obtained with 550 mV, and 85 % COD consumption was attained. No considerable differences in the hydrogen production rate were observed when the COD was increased from 400 to 1200 mg/L using 550 mV. However, maximal hydrogen production rates were obtained with the lower COD concentration using 350 mV. Neither the employment of AEM and CEM nor the change from synthetic substrate to real substrate resulted in remarkable changes in MEC performance. The substrate containing glucose was more slowly degraded because glucose was first transformed into VFAs, and then the VFAs were consumed to produce hydrogen. In this case, methane and carbon dioxide were detected.