This study evaluated the effect of repeated heat treatment towards the enhancement of hydrogen fermentation from galactose in an upflow anaerobic sludge blanket reactor with the hydraulic retention time of 6 h and the operation temperature of 37 degrees C. The hydrogen production rate (HPR) and hydrogen yield (HY) gradually increased up to 9.1 L/L/d and 1.1 mol/mol galactose, respectively, until the 33rd day of operation. When heat treatment at 80 degrees C for 30 min was applied, hydrogen production performance was enhanced by 37% with the enrichment of hydrogen producing bacteria population. The HPR and HY were achieved at 12.5 L/L/d and 1.5 mol/mol hexose, respectively, during further 30 cycles of reactor operation. The repeated heat treatment would be a viable strategy to warrant reliable continuous hydrogen production using mixed culture. (C) 2017 Elsevier Ltd. All rights reserved.