Factors affecting hydrogen production from the co-digestion of food waste and sludge in batch fermentation by anaerobic mixed cultures were optimized using Response Surface Methodology with Central Composite Design. Investigated parameters included C/N ratio, inoculums concentration, Na(2)HPO(4) concentration and Endo nutrient addition. The experiments were conducted in 120 mL serum bottles with a working volume of 70 mL. Results revealed that the optimum conditions were a C/N ratio of 33.14, inoculums concentration of 2.70 g-VSS/L, Na(2)HPO(4) concentration of 6.27 g/L and Endo nutrient addition of 7.51 mL/L. Under the optimal conditions, a maximum hydrogen yield (HY) of 102.63 mL H(2)/g-VS(added) and specific hydrogen production rate (SHPR) of 59.62 mL H(2)/g-VSS h were obtained. C/N ratio and inoculums concentration showed the greatest individual and interactive effects on HY and SHPR (P < 0.05). Endo nutrient addition also had an individual effect on SHPR (P = 0.0124). The confirmation experiment under optimal condition showed an HY and SHPR of 101.14 mL H(2)/g-VS(added) and 59.43 mL H(2)/g-VSS h, respectively. This was only 1.01% and 1.00%, respectively, different from the predicted values. Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.