The paper evaluated the impact of substrate storage on the respirometric assessment of process stoichiometry based on oxygen uptake rate (OUR) measurements. Two parallel sequencing batch reactors were operated with pulse feeding of synthetic substrate mixture at a sludge age of 8 days and 2 days. During the cycle experiments with acetate, 40-45% of acetate was converted to polyhydroxybutyrate, which was partly consumed during each cycle. Respirometric analysis also yielded OUR profiles for the corresponding cyclic operation. A mass balance expression was derived based on oxygen utilization. Oxygen demands calculated on the basis of partial PHB utilization closely matched the experimental values retrieved from OUR profiles within limits of analytical precision. The relative contribution of storage mechanism represented more than 50% of overall oxygen demand. Substrate storage, when totally disregarded or not properly evaluated, was observed to involve an error of around 10% on overall the oxygen demand. (C) 2014 Elsevier Ltd. All rights reserved.