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

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 h) 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 = 0.566) 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 > 0.600). Matrix-wide analyses indicated high consistency in connectivity rankings (Kendall’s W = 0.798).

Among the 23 most temporally stable regions, five connections showed systematic associations with reactive aggression across sessions; none survived FDR correction, consistent with the non-aggressive nature of the cohort. 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 importantly, reliability estimates from this healthy homogeneous sample represent upper-bound values not directly transferable to clinical populations, where greater symptom heterogeneity and state fluctuations are expected to reduce temporal stability.

Within these constraints, identifying stable connectivity patterns through reliability-informed frameworks provides a proof-of-concept for future individual-differences research, while regions with poor reliability require methodological optimization before serving as measurement targets. This research was partially supported by grant number PID2022-142287OA-I00; funded by MCIN/AEI/https://doi.org/10.13039/501100011033 and by ERDF, EU” and The Prometeo Program for research groups of excellence of the Ministry of Innovation, Universities, Science and Digital Society of the Generalitat Valenciana, grant number CIPROM/2021/46. Biomedical Imaging Research Group (GIBI230), La Fe Health Research Institute, Avenida Fernando Abril Martorell, 106, 46026, Valencia, Spain Maria Beser-Robles, Leonor Cerdá‑Alberich & Luis Martí‑Bonmatí Radiology Department, Hospital Universitario y Politécnico La Fe, Valencia, Spain Department of Psychobiology, University of Valencia, Valencia, Spain Carolina Sarrate‑Costa, Luis Moya‑Albiol & Ángel Romero‑Martínez The authors declare no competing interests.

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