Using pH values, temperature, and dissolved oxygen as the influencing factors, a decaying characteristics experiment of activated sludge was carried out by combining the LIVE/DEAD (R) Baclight technique with the 2,3,5-triphenyl tetrazolium chloride - dehydrogenase activity determination method. Using batch experiments, a response surface methodology was applied in the experimental design to determine the most important influential factor in the decay of activated sludge. The activated sludge mixed liquor for the experiment was generated in a laboratory-scale circular plug-flow reactor, which has already been approved for an invention patent. The analyzed results revealed that the most important influential factor in sludge activity decay is the pH, followed by temperature and then dissolved oxygen. After the decay experiment, 40.94-90.03% of sludge activity decay is caused by reduced cell activity, and the rest is due to cell death. (C) 2014 Elsevier Ltd. All rights reserved.