Background: Longitudinal changes in child and adolescent active school transport (AST), and the mediating role of different intensities of daily physical activity (PA) levels in relation to AST and physical fitness and adiposity indicators is unclear. This study aimed to: 1) describe longitudinal changes in AST, light PA (LPA), moderate-to vigorous-intensity PA (MVPA), physical fitness and adiposity indicators over three time-points; and 2) investigate the mediating role of LPA and MVPA levels on associations between AST and physical fitness and adiposity indicators over three time-points among children and adolescents. Methods: This longitudinal study comprised 1646 Spanish children and adolescents (48.8% girls, mean age 12.5 years ±2.5) at baseline, recruited from schools in Cádiz and Madrid. Mode of commuting to school was selfreported at baseline (T0, 2011-12), 1-year (T1) and 2-year follow-up (T2). PA was assessed using accelerometers. Handgrip strength, standing long jump and cardiorespiratory fitness (CRF) assessed physical fitness. Height, weight, body mass index, waist circumference, and triceps and subscapular skinfold thickness were measured. Multilevel linear regression analyses assessed changes in AST, PA levels, physical fitness and adiposity indicators over three time-points (T0-T1-T2). Additionally, longitudinal path analysis (n = 453; mean age [years] 12.6 ± 2.4) was used to test the mediating effects of LPA and MVPA levels on the association between AST and physical fitness and adiposity indicators.