Background and Objectives:While there is growing evidence that physical activity promotes neuronal health, studies examining the relation between physical activity and brain morphology remain inconclusive. We therefore examined whether objectively-quantified physical activity is related to brain volume, cortical thickness and grey matter density in a large cohort study. Additionally, we assessed molecular pathways that may underlie the effects of physical activity on brain morphology.Methods:We used cross-sectional baseline data from 2,550 eligible participants (57.6% women; mean age: 54.7 years, range: 30–94 years) of a prospective cohort study. Physical activity dose (metabolic equivalent hours and step counts) and intensity (sedentary, light-intensity and moderate-to-vigorous intensity activities) were recorded with accelerometers. Brain volumetric, grey matter density and cortical thickness measures were obtained from 3T MRI scans using FreeSurfer and Statistical Parametric Mapping. The relation of physical activity (independent variable) and brain structure (outcome) was examined with polynomial multivariable regression, while adjusting for age, sex, intracranial volume, education and smoking. Using gene expression profiles from the Allen Brain Atlas, we extracted molecular signatures associated with the effects of physical activity on brain morphology.Results:Physical activity dose and intensity were independently associated with larger brain volumes, grey matter density and cortical thickness of several brain regions. The effects of physical activity on brain volume were most pronounced at low physical activity quantities and differed between men and women and across age. For example, more time spent in moderate-to-vigorous intensity activities was associated with greater total grey matter volume, but the relation leveled off with more activity (standardized ß [95% confidence intervals]: 1.37 [0.35, 2.39] and -0.70 [-1.25, -0.15] for the linear and quadratic terms, respectively). The strongest effects of physical activity were observed in motor regions and cortical regions enriched for genes involved in mitochondrial respiration.Discussion:Our findings suggest that physical activity benefits brain health, with the strongest effects in motor regions and regions with a high oxidative demand. While young adults may particularly profit from additional high-intensity activities, older adults may already benefit from light-intensity activities. Physical activity and reduced sedentary time may be critical in the prevention of age-associated brain atrophy and neurodegenerative diseases.