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Background: Increasing physical activity (PA) is an effective strategy to slow reductions in cortical volume and maintain cognitive function in older adulthood. However, PA does not exist in isolation, but coexists with sleep and sedentary behaviour to make up the 24-hour day. We investigated how the balance of all three behaviours (24-hour time-use composition) is associated with grey matter volume in healthy older adults, and whether grey matter volume influences the relationship between 24-hour time-use composition and cognitive function. Methods: This cross-sectional study included 378 older adults (65.6 ± 3.0 years old, 123 male) from the ACTIVate study across two Australian sites (Adelaide and Newcastle). Time-use composition was captured using 7-day accelerometry, and T1-weighted magnetic resonance imaging was used to measure grey matter volume both globally and across regions of interest (ROI: frontal lobe, temporal lobe, hippocampi, and lateral ventricles). Pairwise correlations were used to explore univariate associations between time-use variables, grey matter volumes and cognitive outcomes. Compositional data analysis linear regression models were used to quantify associations between ROI volumes and time-use composition, and explore potential associations between the interaction between ROI volumes and time-use composition with cognitive outcomes. Results: After adjusting for covariates (age, sex, education), there were no significant associations between time-use composition and any volumetric outcomes. There were significant interactions between time-use composition and frontal lobe volume for long-term memory (p = 0.018) and executive function (p = 0.018), and between time-use composition and total grey matter volume for executive function (p = 0.028). Spending more time in moderate-vigorous PA was associated with better long-term memory scores, but only for those with smaller frontal lobe volume (below the sample mean). Conversely, spending more time in sleep and less time in sedentary behaviour was associated with better executive function in those with smaller total grey matter volume. Conclusions: Although 24-hour time use was not associated with total or regional grey matter independently, total grey matter and frontal lobe grey matter volume moderated the relationship between time-use composition and several cognitive outcomes. Future studies should investigate these relationships longitudinally to assess whether changes in time-use composition correspond to changes in grey matter volume and cognition. [ABSTRACT FROM AUTHOR]