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Background  Neuromelanin‐sensitive MRI (NM‐MRI) of the substantia nigra provides a noninvasive way to acquire an indirect measure of dopamine functioning. Despite the potential of NM‐MRI as a candidate biomarker for dopaminergic pathology, studies about its reproducibility are sparse.    Purpose  To assess the test–retest reproducibility of three commonly used NM‐MRI sequences and evaluate three analysis methods.    Study Type  Prospective study.    Population  A total of 11 healthy participants age between 20–27 years.    Field Strength/Sequence  3. 0T ;  NM‐MRI  gradient recalled echo ( GRE ) with magnetization transfer ( MT ) pulse;  NM‐MRI  turbo spin echo ( TSE ) with  MT  pulse;  NM‐MRI TSE  without  MT  pulse.    Assessment  Participants were scanned twice with a 3‐week interval. Manual analysis, threshold analysis, and voxelwise analysis were performed for volume and contrast ratio (CR) measurements.    Statistical Tests  Intraclass correlation coefficients (ICCs) were calculated for test–retest and inter‐ and intrarater variability.    Results  The GRE sequence achieved the highest contrast and lowest variability (4.9–5.7%) and showed substantial to almost perfect test–retest ICC (0.72–0.90) for CR measurements. For volume measurements, the manual analysis showed a higher variability (10.7–17.9%) and scored lower test–retest ICCs (–0.13–0.73) than the other analysis methods. The threshold analysis showed higher test–retest ICC (0.77) than the manual analysis for the volume measurements.    Data Conclusion  NM‐MRI is a highly reproducible measure, especially when using the GRE sequence and CR measurements. Volume measurements appear to be more sensitive to inter/intrarater variability and variability in placement and orientation of the NM‐MRI slab. The threshold analysis appears to be the best alternative for volume analysis.    Level of Evidence  2    Technical Efficacy Stage  1

Reliability and Reproducibility of Neuromelanin‐Sensitive Imaging of the Substantia Nigra: A Comparison of Three Different Sequences