Test-retest reliability of resting-state functional connectivity: a methodological investigation with implications for aggression research

Abstract The temporal stability of resting-state functional MR connectivity (rsfMRc) is a fundamental methodological consideration for longitudinal neuroimaging research, yet systematic evaluations across clinically relevant time frames remain limited. This study examined test-retest reliability of rsfMRc over short-term (1 hour) and medium-term (1 month) intervals in 34 healthy young adult males across three sessions. Intraclass correlation coefficients (ICC) and matrix-wide reliability metrics were calculated to evaluate temporal stability across major brain networks, and associations between stable connectivity patterns and aggression measures were examined. Overall reliability showed moderate values (mean ICC = .557) with significant network-specific variation. Salience Network regions, particularly the supramarginal gyrus, and lateral parietal regions of the Default Mode Network demonstrated the highest stability (ICC > .600). Matrix-wide analyses indicated high consistency in connectivity rankings (Kendall's W = .798). Among the 23 most temporally stable regions, several showed systematic associations with both reactive and proactive aggression. These findings reveal substantial heterogeneity in rsfMRc reliability across brain networks and measurement intervals, providing empirical guidance for researchers planning longitudinal connectivity studies. Network-specific considerations are critical in study design, and identifying stable connectivity patterns offers a foundation for biomarker development, while regions with poor reliability may require alternative acquisition strategies.