Considerable research has been devoted in the past decades to the optimization and control of biological wastewater treatment processes. Many treatment processes have been studied to increase the methane potential of sludge with a rate limiting hydrolysis stage of organic matters associated with microbial cells. Although numerous informations are currently available about sludge minimization processes in WWTP, as sonication, few data are available about its fundamental mechanisms especially microbial changes. In order to clarify the relationship between sludge reduction efficiency and both chemical and biological modifications, the effects of sonication on activated sludge were investigated by combining the monitoring of cell lysis, organic matter solubilization, floc structure and biodegradability. So the aim of this study was to characterize the effects of activated sludge sonication, in terms of cell lysis, organic matter solubilization and biodegradability. For this, in parallel to the organic matter solubilization degree determination (Chemical Oxygen Demand (COD), proteins and sugars), cell disruption in activated sludge was monitored with Flow Cytometry (FCM) before and after sonication. Prior to FCM analyses, activated sludge samples were disaggregated, filtered and stained with a DNA specific fluorescent dye, the Sytox Green, which diffuses only damaged cells. Structural changes during sonication were assessed by microscopic observations and granulometry. Finally, the real impact of thermal treatment on sludge biodegradability was studied under anaerobic conditions. The results showed that, for a specific energy ranging from 0 to 210000 kJ/kg TS, sonication did not induce cell lysis since green fluorescence intensity remained approximately constant before and after sonication. On the contrary, COD, sugars and proteins solubilization degrees reach 27%, 16% and 18% respectively. These results, that showed a progressive release of organic matter particulate components without cell disruption, suggested that the origin of the organic matter released after sonication was only extracellular, probably due to the polymeric network disruption. Moreover, the results from anaerobic biodegradability tests did not show any improvement after sonication, indicating that the specific energy was not high enough to increase anaerobic biodegradability of the sludge. Even if sonication induced a progressive release of organic matter components from particulate to soluble fraction, the specific energy applied was not high enough to induce cell lysis, and consequently, anaerobic biodegradability improvement. Moreover, this study showed that hydrolysis treatments improvement requires a precise knowledge of both biological and chemical changes induced during hydrolysis processes.