Thermophilic (55 degrees C) mixed culture fermentation (MCF) is a promising technology for converting organic wastes into bioproducts including hydrogen and chemicals. In this study, the effect of pH, hydrogen partial pressure (P-H2) and influent glucose concentration on the metabolic distribution in thermophilic chemostat was investigated. The results showed that the metabolite composition was changed from acetate, butyrate and hydrogen to acetate, ethanol, propionate and format when pH increased from 4.0 to 7.0. The conversion of hydrogen to formate was found to be thermodynamically controlled. Reducing P-H2 did not significantly increase the hydrogen yield and consequently the metabolites did not change much. Lactate accumulation was observed only at high influent glucose concentration. The maximum hydrogen yield was 2.2 +/- 0.16 mol/mol-glucose at P-H2 0.05 atm, while the maximum hydrogen production rate was 7.9 +/- 0.1 L/(L-reactor.Day) at influent glucose concentration of 76.3 mmol/L. Thus, considering the operating cost, increasing influent glucose concentration to 76.3 mmol/L in this work could be a more practical approach to produce hydrogen in thermophilic MCF. Meanwhile, the impacts of precedent pH and influent glucose concentration on the metabolites distribution were negligible in thermophilic chemostat. The thermodynamic analysis illustrated that a mixture of acetate, butyrate, ethanol, propionate and/or lactate was essential for thermophilic hydrogen producing MCF. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.