Dark fermentation experiments were performed for bio-hydrogen production from ground wheat starch solution (10 +/- 1 g l(-1)) using periodic feeding and effluent removal. A mixed culture of Clostridium butyricum-NRRL 1024 and Clostridium pasteurianum-NRRL B-598 were used with an initial biomass ratio of 1/1. Effects of wheat starch loading rate on the rate and yield of bio-hydrogen formation were investigated. Substrate loading rate was varied between 0.54 and 5.52 g d(-1) (HRT = 6-60 h). The highest hydrogen formation rate (280 ml d(-1)), volumetric hydrogen formation rate (1857 ml H(2) l(-1) d(-1)) and volatile fatty acids (VFAs) concentration were obtained with a substrate loading rate of 5.52 g d(-1) (HRT = 6 h). The highest hydrogen yield (109 ml H(2) g TS(-1)) was obtained with a substrate loading rate of 1.38 g d(-1). Copyright (C) 2011, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.