This study investigated the effects of sugar composition and hydraulic retention time (HRT) on continuous hydrogen fermentation. Continuously-stirred tank reactors (CSTRs) were inoculated with heat-treated digester sludge and fed with 15 g/L of glucose, galactose and galactose: glucose mixture (8:2 ratio-simulating the hydrolysate composition of macroalgae) at HRTs of 6-24 h. Peak hydrogen production rate (HPR) and hydrogen yield (HY) of 4.49 L/L/d and 1.62 mol/mol glucose(added) were attained while using glucose as feedstock at HRTs of 6 and 18 h, respectively. Meanwhile, galactose provided a peak HPR and HY of 2.35 L/L/d and 1.00 mol/mol galactose(added) during the HRTs of 12 and 24 h, respectively. In case of mixed sugars (galactose 8: glucose 2) the production performances were inferior to the sole sugar conditions due to the low substrate utilization of less than 65%, which showed a maximal HPR and HY of 2.75 L/L/d and 0.48 mol/mol carbohydrate(added) at the HRTs of 6 and 18 h, respectively. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.