To assess the effect of time-of-day (TOD) on automated measures of brain morphometry derived from T1-weighted (T1W) images.A recent study reported subtle, yet significant changes in total brain volume (TBV) from morning to evening in an exceptionally large group of patient populations as well as healthy elderly individuals [3]. However, it is unknown whether: (a) TOD can influence measures of brain morphometry derived from T1W images in studies involving relatively smaller groups of young healthy individuals; (b) TOD may differentially affect the apparent volume of the three main tissue compartments (gray matter (GM); white matter (WM), and cerebrospinal fluid (CSF) (c) specific brain structures and cortical regions are disproportionately impacted; (d) the apparent change in TBV impacts surface-based T1W morphometry measures (e.g. cortical thickness) as well as Voxel-Based-Morphometry (VBM) measures.A group of 19 healthy young adults (mean age: 25.8 years, range: 20-38, 10 female) were scanned during two visits (each visit scheduled two weeks apart) on a 3 Tesla GE MR750 scanner using a GE 32-channel head coil. During both visits, participants were scanned from 10AM to 12PM and from 2PM to 4PM, with a two-hour rest period in between. Two T1W MPRAGE volumes were acquired during each session (scan parameters: accelerated sagittal 3D MPRAGE, 25 cm FOV, 256x256 matrix slice, 1 mm slice thickness, TR: 7 ms, TE: 3.42 ms, flip angle: 7 deg. For the volumetric and surface-based analysis, we used the automated longitudinal pipeline implemented in FreeSurfer (version 5.3) to build subject-specific templates, and each participant’s images were re-processed using information from their subject-specific template⁴ to derive various volumetric and surface-based metrics. All volumetric measurements were expressed as a percentage of intra cranial volume (ICV), which was automatically estimated by FreeSurfer. Surface-based measures, such as cortical thickness and surface area as well as indices of brain curvature were automatically extracted using FreeSurfer’s automatic reconstruction procedure and the spatial topography of the TOD effect on different cortical regions was investigated using an automatic parcellation procedure [5]. For the VBM analysis⁶ (FSL-VBM version 1.0), all T1W images were processed through a modified VBM-FSL pipeline designed for longitudinal analysis⁷. To test the relationship between each brain morphometry measure and TOD, we applied the linear mixed effect (LME) model using the lme4 package in R⁸. For each measure of brain morphometry, the model used consisted of fixed effects (i.e. TOD) for both intercept and slope and a random effect (i.e., the subject-specific morphometric measure) to account for individual variations and estimate a prediction for each value of the T1W structural measure being modeled. The residual error between the prediction and actual measurement was calculated and used for significance testing.Our results reveal that between morning and afternoon: (a) A highly significant reduction in apparent brain volume (1.145% of ICV/12 h) can be detected even in a small group of adult healthy volunteers (b) The apparent volume of all the three major tissue compartments, GM, WM and CSF are influenced, and the magnitude of the TOD effect varies across the tissue compartments (Fig. 1); (c) The increase in CSF volume was associated with the decrease in GM and WM volumes; (d) Measures of apparent cortical thickness, surface area and GMD were all affected by TOD (Fig. 2), while measures such as, curvature indices and sulcal depth were not impacted; (e) The effect of TOD appears to have a greater impact on morphometric measures of the frontal and temporal lobe compared to other major lobes of the brain (Fig. 2). Our findings suggest that automatically derived measures of brain morphometry that are thought to reflect properties such as cortical thickness, surface area, GM volume and GMD, change significantly from morning to afternoon, even in the absence of any experimental manipulation. The magnitude of the TOD related changes in measures such as cortical thickness is greater than the apparent change attributed to interventions, such as memory training⁹. Moreover, the spatial topography of the TOD effect on apparent cortical thickness and GMD indicate that the frontal and temporal lobes are disproportionately impacted. This suggests that controlling for the effect of TOD is crucial for proper interpretation of apparent structural differences measured using T₁W morphometry with longitudinal and cross-sectional experimental designs. The strong correlation between changes in CSF volume and changes in GM and WM volumes from morning to afternoon, as well as reports of similar changes in the literature¹⁰ suggests that the TOD effect is a physiological phenomenon that warrants further investigation.