While physical and mental health benefits of regular physical activity are well known, increasing evidence suggests that limiting sedentary behaviour is also important for health. Evidence shows associations of physical activity and sedentary behaviour with health-related quality of life (HRQoL), however, these findings are based predominantly on duration measures of physical activity and sedentary behaviour (e.g., minutes/week), with less attention on frequency measures (e.g., number of bouts). We examined the association of HRQoL with physical activity and sedentary behaviour, using both continuous duration (average daily minutes) and frequency (average daily bouts≥10 min) measures. Baseline data from the WALK 2.0 trial were analysed. WALK 2.0 is a randomised controlled trial investigating the effects of Web 2.0 applications on engagement, retention, and subsequent physical activity change. Daily physical activity and sedentary behaviour (duration = average minutes, frequency = average number of bouts ≥10 minutes) were measured (ActiGraph GT3X) across one week, and HRQoL was assessed with the ‘general health’ subscale of the RAND 36-Item Health Survey. Structural equation modelling was used to evaluate associations. Participants (N = 504) were 50.8±13.1 (mean±SD) years old with a BMI of 29.3±6.0. The 465 participants with valid accelerometer data engaged in an average of 24.0±18.3 minutes and 0.64±0.74 bouts of moderate-vigorous physical activity per day, 535.2±83.8 minutes and 17.0±3.4 bouts of sedentary behaviour per day, and reported moderate-high general HRQoL (64.5±20.0). After adjusting for covariates, the duration measures of physical activity (path correlation = 0.294, p<0.05) and sedentary behaviour were related to general HRQoL (path coefficient = -0.217, p<0.05). The frequency measure of physical activity was also significant (path coefficient = -0.226, p<0.05) but the frequency of sedentary behaviour was not significantly associated with general HRQoL. Higher duration levels of physical activity in fewer bouts, and lower duration of sedentary behaviour are associated with better general HRQoL. Further prospective studies are required to investigate these associations in different population groups over time.