Nowadays, hydrogen produced globally has been synthesized from fossil fuel with limited source. Therefore, research has been developed in order to explore biological H2 production by dark fermentation. The purpose of this work was to evaluate the effect of initial pH and ferrous sulfate and ammonium sulfate concentrations on the production of biohydrogen by dark fermentation. The process was carried out in batch mode under anaerobic conditions, in the absence of light, and at standard room temperature and pressure. A microbial consortium provided by the effluent treatment plant of a local dairy company was inoculated into a synthetic medium supplemented with cheese whey permeate (20 g/L of lactose) as a carbon source. The influence of three variables was analyzed by a central composite design 2((3)), and the optimum results of hydrogen yield (4.13 mol H2/mol lactose) and productivity (86.31 mmol H2/L/day) were achieved at initial pH 7.0 and FeSO4 and (NH4)2SO4 concentrations of 0.6 and 1.5 g/L, respectively. Under these conditions, the kinetic parameters of fermentation were investigated by analyzing the profile of H2 yield and productivity, metabolite concentrations, pH, and concentration of dissolved iron. In the kinetic analysis, the modified Gompertz equation described adequately the fermentative hydrogen production from cheese whey permeate (R (2) = 0.98). The profile of ethanol and volatile organic acids showed that lactic acid and butyric acid were the main metabolites produced, and the sum of both by-products corresponded to about 58 % of the total metabolites.