BACKGROUND: To examine if a composite activity-sleep behaviour index (ASI) mediates the effects of a combined physical activity and sleep intervention on symptoms of depression, anxiety, or stress, quality of life (QOL), energy and fatigue in adults. METHODS: This analysis used data pooled from two studies: Synergy and Refresh. Synergy: Physically inactive adults (18-65 years) who reported poor sleep quality were recruited for a two-arm Randomised Controlled Trial (RCT) (Physical Activity and Sleep Health (PAS; n = 80), or Wait-list Control (CON; n = 80) groups). Refresh: Physically inactive adults (40-65 years) who reported poor sleep quality were recruited for a three-arm RCT (PAS (n = 110), Sleep Health-Only (SO; n = 110) or CON (n = 55) groups). The SO group was omitted from this study. The PAS groups received a pedometer, and accessed a smartphone/tablet "app" using behaviour change strategies (e.g., self-monitoring, goal setting, action planning), with additional email/SMS support. The ASI score comprised self-reported moderate-to-vigorous-intensity physical activity, resistance training, sitting time, sleep duration, efficiency, quality and timing. Outcomes were assessed using DASS-21 (depression, anxiety, stress), SF-12 (QOL-physical, QOL-mental) and SF-36 (Energy & Fatigue). Assessments were conducted at baseline, 3 months (primary time-point), and 6 months. Mediation effects were examined using Structural Equation Modelling and the product of coefficients approach (AB), with significance set at 0.05. RESULTS: At 3 months there were no direct intervention effects on mental health, QOL or energy and fatigue (all p > 0.05), and the intervention significantly improved the ASI (all p < 0.05). A more favourable ASI score was associated with improved symptoms of depression, anxiety, stress, QOL-mental and of energy and fatigue (all p < 0.05). The intervention effects on symptoms of depression ([AB; 95%CI] -0.31; - 0.60,-0.11), anxiety (- 0.11; - 0.27,-0.01), stress (- 0.37; - 0.65,-0.174), QOL-mental (0.53; 0.22, 1.01) and ratings of energy and fatigue (0.85; 0.33, 1.63) were mediated by ASI. At 6 months the magnitude of association was larger although the overall pattern of results remained similar. CONCLUSIONS: Improvements in the overall physical activity and sleep behaviours of adults partially mediated the intervention effects on mental health and quality of life outcomes. This highlights the potential benefit of improving the overall pattern of physical activity and sleep on these outcomes. TRIAL REGISTRATION: Australian New Zealand Clinical Trial Registry: ACTRN12617000680369 ; ACTRN12617000376347 . Universal Trial number: U1111-1194-2680; U1111-1186-6588. Human Research Ethics Committee Approval: H-2016-0267; H-2016-0181.