Diurnal Variations and Test-Retest Reliability of Resting-State Functional MRI Metrics

Resting-state fMRI (rs-fMRI) is widely used to assess intrinsic brain activity, yet concerns about its test-retest reliability and reproducibility persist. Circadian rhythms strongly influence brain physiology, but their impact on rs-fMRI reliability remains poorly understood. In this study, we scanned 39 healthy young adults six times within a single day (08:00-20:00) under standardized conditions. For each session, we computed four common rs-fMRI metrics, including amplitude of low-frequency fluctuations (ALFF), wavelet-transformed ALFF (wALFF), fractional ALFF (fALFF), and regional homogeneity (ReHo), and assessed reliability using intraclass correlation coefficients (ICCs). ReHo showed relatively higher and more stable reliability across sessions, whereas amplitude-based metrics, particularly fALFF, exhibited greater diurnal variation. Both network-level and region-specific analyses revealed low reliability in the limbic and subcortical structures, with a mid-morning dip at 10:00. Moreover, ICCs for ALFF, wALFF, and fALFF declined with increasing inter-scan intervals, whereas ReHo remained robust. These findings demonstrate diurnal fluctuations in rs-fMRI reliability, with different metrics exhibiting distinct temporal stability profiles. We recommend that scan timing and circadian influences should be explicitly considered in the design, analysis, and interpretation of future rs-fMRI studies.