ObjectiveTo examine changes in the prevalence of six key chronic disease risk factors (the “Big 6”), from before (2019) to during (2021) the COVID-19 pandemic, among a large and geographically diverse sample of adolescents, and whether differences over time are associated with lockdown status and gender.DesignProspective cohort study.SettingThree Australian states (New South Wales, Queensland and Western Australia) spanning over 3000 km.Participants983 adolescents (baseline M_age=12.6, SD=0.5, 54.8% girl) drawn from the control group of the Health4Life Study.Primary outcomesThe prevalence of physical inactivity, poor diet (insufficient fruit and vegetable intake, high sugar-sweetened beverage intake, high discretionary food intake), poor sleep, excessive recreational screen time, alcohol use and tobacco use.ResultsThe prevalence of excessive recreational screen time (prevalence ratios (PR)=1.06, 95% CI=1.03 to 1.11), insufficient fruit intake (PR=1.50, 95% CI=1.26 to 1.79), and alcohol (PR=4.34, 95% CI=2.82 to 6.67) and tobacco use (PR=4.05 95% CI=1.86 to 8.84) increased over the 2-year period, with alcohol use increasing more among girls (PR=2.34, 95% CI=1.19 to 4.62). The prevalence of insufficient sleep declined across the full sample (PR=0.74, 95% CI=0.68 to 0.81); however, increased among girls (PR=1.24, 95% CI=1.10 to 1.41). The prevalence of high sugar-sweetened beverage (PR=0.61, 95% CI=0.64 to 0.83) and discretionary food consumption (PR=0.73, 95% CI=0.64 to 0.83) reduced among those subjected to stay-at-home orders, compared with those not in lockdown.ConclusionLifestyle risk behaviours, particularly excessive recreational screen time, poor diet, physical inactivity and poor sleep, are prevalent among adolescents. Young people must be supported to find ways to improve or maintain their health, regardless of the course of the pandemic. Targeted approaches to support groups that may be disproportionately impacted, such as adolescent girls, are needed.Trial registration numberAustralian New Zealand Clinical Trials Registry (ACTRN12619000431123)