► Micro-tome slicing technique, when combined with other tools, is a promising tool to provide a spatial distribution of physical, chemical and microbiological structure of cake layers. ► Cake layers have a loose outer surface when compared to bottom layer. ► Physical and chemical structure change significantly along the depth of cake layer. ► Microbial population density changes along the cake layer depth. A laboratory-scale submerged anaerobic membrane bioreactor (SAnMBR) was used for thermomechanical pulping whitewater treatment. Sludge cake formation on membrane surfaces was identified as the dominant mechanism of membrane fouling. The spatial distribution of physical, chemical and microbiological structure of cake layers was characterized by various analytical techniques, including micro-tome slicing technique, confocal laser scanning microscopy (CLSM), conventional optical microscopy (COM), scanning electron microscopy (SEM)-energy-dispersive X-ray analyzer (EDX), particle size distribution (PSD) analysis, Fourier transform infrared (FTIR) spectroscopy, extraction and chemical analysis of extracellular polymeric substances (EPS), and polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (DGGE). The results showed that the areal porosity decreased from the top layer to the bottom layer. Smaller flocs had a higher tendency to accumulate on membrane surfaces but the consolidation of cake sludge in the bottom layers resulted in larger flocs as compared to the fresh cake sludge formed on the top layers. There was an increase in the bound EPS density (mg EPS/cm 3 wet sludge) and a decrease in the ratio of proteins to polysaccharides in bound EPS from the top to bottom layers. PCR-DGGE study showed that there were significant differences in microbial community population density along the cake layer depth. Through the CLSM and COM images, cake layer was found to have a loose outer surface when compared with the cake bottom. The results provide a new insight in cake layer structure and suggest that structures change significantly from the top layer to the bottom layer.