In this study, two experiments were conducted to evaluate the impact of substrate concentrations on methane potential and degradation kinetics of substrate. The biochemical methane potential (BMP) tests in Experiment I were performed at a constant inoculum to substrate ratio (ISR), whereas, different ISRs were applied in Experiment II. Results obtained from Experiment I revealed that methane potential of substrate increased at a saturating trend with higher substrate concentrations, and could differ by up to 30% between the lowest and highest investigated concentrations. The results of Experiment II verified the results of Experiment I, and further showed that this trend also occurs when the substrate concentration is regulated with ISRs. In contrast, substrate concentration had no significant impact on the degradation kinetics. It was concluded that dilutions should be avoided when the substrate concentration is lower than 10 g VS/L in order to avoid underestimations of methane potential from BMP test. (C) 2015 Elsevier Ltd. All rights reserved.