Anaerobically digested sludge was seeded in a mesophilic UASB reactor and the sludge was monitored for seven months to better understand the start-up process of the reactor. The reactor was fed with synthetic wastewater containing glucose. As the COD loading rate increased stepwise (from 1 to 4 g COD l-1 d-1), the methane production rate increased. COD removal efficiency was maintained to be greater than 90% after day 36. Maximum value of the methane production rate (6.0-6.5 l d-1) was achieved from day 152 and remained stable afterward. Although the reactor showed steady performance in terms of COD removal efficiency and methane production under constant hydraulic retention time (HRT) or COD loading rates, physicochemical and microbial properties of UASB sludge kept changing during the initial 5 months of operation. Specific methanogenic activity was initially negligible but increased until day 150, and then remained constant (0.72 + 0.11 g CH4-COD g-1 VSS d-1) afterward. Sludge volume index showed that the settling ability of UASB sludge gradually improved until it reached a plateau in day 120. Improved settling-ability could provide a basis for keeping bed height constant despite shortened HRT. The mean diameter of the UASB sludge gradually increased until approximately day 150 and maintained a maximum value (0.48 mm) afterward. Confocal laser scanning microscopy revealed F420-based autofluorescence of physical and optical sections of UASB sludge, suggesting the locations of autofluorescent methanogens in the UASB sludge during the start-up period. During the initial operation of the reactor, autofluorescence showed random and uneven distribution inside the sludge. However, autofluorescence appeared as an inner layer near the edge of the sludge with time, suggesting more abundant or active methanogens in this layer. The highest autofluorescence was observed in the range of 20 to 28 µm depth from the surface of granule as determined by optical slicing of UASB sludge. The results obtained in this study provide insight into UASB sludge development that involves dynamic changes in physicochemical and microbial aspects during the start-up period.