INTRODUCTION: Several astronauts have experienced severe headaches during spaceflight, but no studies have examined the associated brain microstructure and functional changes. Head-down-tilted bed rest (HDBR) is a well-established method for studying the physical effects of microgravity on the ground. In this study, we analyzed the changes in brain microstructure and function during headache caused by HDBR using diffusion tensor imaging (DTI) and resting state functional magnetic resonance imaging (R-fMRI). METHODS: We imaged 28 healthy subjects with DTI and R-fMRI in the horizontal supine position and HDBR. Using Tract-Based Spatial Statistics, fractional anisotropy, mean diffusivity, radial diffusivity, and axial diffusivity were compared between the headache and non-headache groups. Additionally, an analysis of functional connectivity (FC) was performed, followed by a correlation analysis between FC and numerical rating scale. RESULTS: HDBR caused headaches in 21 of 28 subjects. DTI analysis showed no significant change in fractional anisotropy after HDBR, whereas axial diffusivity, radial diffusivity, and mean diffusivity increased significantly. R-fMRI analysis showed a significant decrease in FC in several areas after HDBR. The headache group showed significantly higher FC before HDBR, and both groups showed higher FC after HDBR. Correlation analysis showed a positive correlation between FC and numerical rating scale before HDBR but negative after HDBR. DISCUSSION: We demonstrated the image change in the acute phase of space headache by HDBR using DTI and R-fMRI. Changes in brain microstructure and function specific to patients developing headaches may be evaluated by imaging. Goto M, Shibata Y, Ishiyama S, Matsumaru Y, Ishikawa E. Brain microstructure and brain function changes in space headache by head-down-tilted bed rest . Aerosp Med Hum Perform. 2023; 94(9):678-685.