Hydrogen production from cheese whey through dark fermentation was investigated in this study in order to systematically analyse the effects of the operating pH. The effluents from pecorino cheese and mozzarella cheese production were the substrates used for the fermentation tests. Either CW only or a mixture of CW and heat-shocked activated sludge were used in mesophilic pH-controlled batch fermentation experiments. The results indicated that hydrogen production was strongly affected by multiple factors including the substrate characteristics, the addition of an inoculum as well as the pH. The process variables were found to affect to a varying extent numerous interrelated aspects of the fermentation process, including the hydrogen production potential, the type of fermentation pathways, as well as the process kinetics. The fermentation products varied largely with the operating conditions and mirrored the H-2 yield. Significant fermentative bio-hydrogen production was attained at pHs of 6.5-7.5, with the best performance in terms of H2 generation potential (171.3 NL H-2/kg TOC) being observed for CW from mozzarella cheese production, at a pH value of 6.0 with the heat-shocked inoculum. Copyright (c) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.