The distribution of physical activity bouts through the day may provide useful information for assessing the impacts of interventions on aspects such as physical function. This study aimed to investigate the associations between physical activity fragmentation, tested using different minimum physical activity bout lengths, with physical function in older adults. The SITLESS project recruited 1360 community-dwelling participants from four European countries (≥65 years old). Physical activity fragmentation was represented as the active-to-sedentary transition probability (ASTP), the reciprocal of the average physical activity bout duration measured using ActiGraph wGT3X+ accelerometers. Four minimum bout lengths were utilised to calculate the ASTP: ≥10-s, ≥60-s, ≥120-s and ≥300-s. Physical function was assessed using the 2-min walk test (2MWT) and the composite score from the Short Physical Performance Battery (SPPB) test. Linear regression analyses, after adjusting for relevant covariates, were used to assess cross-sectional associations. After adjustment for relevant covariates, lower ASTP using ≥10-s bouts were associated with longer 2MWT distances and higher SPPB scores. Lower ASTP using ≥120-s bouts and ≥300-s bouts were associated with longer 2MWT distances but not the SPPB. Less fragmented physical activity patterns appeared to be associated with better physical function in community-dwelling older adults.