In this study, the effects of pretreatment temperature and time during conventional and unconventional, microwave-assisted heat shock on the hydrogen producing capability of anaerobic seed sludge from soluble starch was focused. It was found that the different heat transfer techniques resulted in seed cultures with comparable hydrogen production potentials, with the highest obtainable values of approximately 0.9 L H-2/L-d. A comprehensive, statistical analysis revealed that both treatment temperature and time could be designated as significant process variables, however, in distinguishable extents for the two alternative methods. The results indicated that microwave-based sludge pretreatment needed remarkably shorter curing times (2 min) to eliminate H-2-consuming, methanogenic activity in comparison to the conventional heat shock method (30 mm). It was also demonstrated that microwave irradiation increased the soluble organic matter content in the seed sludge. Copyright (C) 2014, Hydrogen Energy Publications, LLC. Published by Elsevier Ltd. All rights reserved.