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A novel method for quantitative measurement of myocardial blood flow (MBF) using arterial spin labeling (ASL) in a single breath-hold is presented, evaluated by simulations, phantom studies and in vivo studies and tested for reproducibility and variability.A flow-sensitive alternating inversion recovery (FAIR) ASL method with Look-Locker readout (LL-FAIR-ASL) was implemented at 3 tesla. Scans were performed on 10 healthy volunteers and MBF measured in three slices. The method was investigated for reproducibility by Bland-Altman analysis and statistical measures, the coefficients of reproducibility (CR) and variation (CV) are reported.The MBF values for the basal, mid, and apical slices were 1.04 ± 0.40, 1.06 ± 0.46, and 1.06 ± 0.38 ml/g/min, respectively (mean ± SD), which compare well with literature values. The CV across all scans, 43%, was greater than the between-session and within-session values, at 16 and 13%, respectively, for the mid-ventricular slice. The change in MBF required for detection, from the CR, was 61% between-session and 53% within-session for the mid-ventricle.This study shows the feasibility of the LL-FAIR-ASL method for the quantification of MBF. The statistical measures reported will allow the planning of future clinical research studies involving rest and stress measurements.