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Background: Greater public transport use has been linked to higher physical activity levels. However, neither the amount of physical activity associated with each daily public transport trip performed, nor the potential total physical activity gain associated with an increase in trips/day, has been determined. Using objective measures, we aimed to quantify the association between public transport use, physical activity and sedentary time. Methods: A longitudinal study of Australian adults living in Hobart, Tasmania, who were infrequent bus users (≥ 18 years; used bus ≤ 2 times/week). The number of bus trips performed each day was determined from objective smartcard data provided by the public transportation (bus) provider across a 36-week study timeframe. Accelerometer measured steps/day (primary outcome), moderate-to-vigorous physical activity (min/day), and sedentary time (min/day) were assessed across four separate one-week periods. Results: Among 73 participants across 1483 day-level observations, on days that public transport was used, participants achieved significantly more steps (β = 2147.48; 95%CI = 1465.94, 2829.03), moderate to vigorous physical activity (β = 22.79; 95% CI = 14.33, 31.26), and sedentary time (β = 37.00; 95% CI = 19.80, 54.21) compared to days where no public transport trips were made. The largest increase in steps per day associated with a one-trip increase was observed when the number of trips performed each day increased from zero to one (β = 1761.63; 95%CI = 821.38, 2701.87). The increase in the number of steps per day was smaller and non-significant when the number of trips performed increased from one to two (β = 596.93; 95%CI=-585.16, 1779.01), and two to three or more (β = 632.39; 95%CI=-1331.45, 2596.24) trips per day. Significant increases in sedentary time were observed when the number of trips performed increased from zero to one (β = 39.38; 95%CI = 14.38, 64.39) and one to two (β = 48.76; 95%CI = 25.39, 72.12); but not when bus trips increased from two to three or more (β=-27.81; 95%CI=-76.00, 20.37). Conclusions: Greater public transport use was associated with higher physical activity and sedentary behaviour. Bus use may yield cumulative increases in steps that amount to 15–30% of the daily recommended physical activity target. A policy and public health focus on intersectoral action to promote public transport may yield meaningful increases in physical activity and subsequent health benefits. [ABSTRACT FROM AUTHOR]