The influence of the soluble microbial products (SMP) and extracellular polymeric substances (EPS) heating extraction method on cell viability was evaluated for each phase of the protocol using epifluorescence microscopy. In addition, the effect of different centrifugation conditions (2700 g at 24 degrees C; 12,000 g at 4 degrees C) was also tested. Sludge samples were collected from a conventional wastewater treatment and a membrane bioreactor (MBR) pilot plant fed in parallel. Results show that different centrifugation parameters do not induce cell membrane damaging. Heating significantly influences membrane integrity; for instance, 75 to 90% of initial viable cells are damaged during this phase, possibly leading to the predominance of protein compared to carbohydrate content. The protein content in EPS is 60 to 88 mg bovine serum albumin/ g volatile suspended solids (VSS); higher values observed in MBR sludge samples are probably attributable to the different characteristics of microbial flocs and process operating parameters. Carbohydrate concentrations are not significantly different regardless of applied procedure and sludge type, and are between 10.4 to 11.6 mg glucose/g VSS.