Frequency-domain optical coherence tomography (FD-OCT) is becoming a useful diagnostic tool for coronary imaging for quantitative coronary analysis. Second-generation FD-OCT produces detailed coronary lumen images. However, the reproducibility of coronary measurements using FD-OCT in humans has not been thoroughly explored. Our goal was to determine the intraobserver, interobserver, and interpullback reproducibility of the in vivo FD-OCT measurements of the lumen area and/or lesion length. Twenty-five patients undergoing coronary angioplasty were included. In all subjects, FD-OCT pullbacks (20 mm/s) were acquired twice from the same coronary segment different from the target lesion, at an interval of 5 minutes, with no other intervention. A total of 9,396 cross-sectional lumen area frames and the relative coronary lesion length of each pullback were analyzed off-line with dedicated software by 2 independent expert readers (A and B). We compared the lumen area and length measurements as follows: pullback 1, read by reader A twice at an interval of 7 days (intraobserver analysis); pullback 1, independently read by readers A and B (interobserver comparison); and pullback 1 versus pullback 2, read by reader A (interpullback comparison). The per-segment and per-frame analyses showed very high and significant correlation coefficients for the interobserver, intraobserver, and interpullback comparisons for the lumen area and lesion length (R ≥0.95 and p <0.001 in all cases). Accordingly, the Bland-Altman estimates of bias showed nonsignificant differences in the interobserver, intraobserver, and interpullback comparisons at all levels, with average biases never >0.150 mm² for the lumen area or 0.200 mm for the lesion length. In conclusion, coronary imaging using FD-OCT showed excellent reproducibility, with low intraobserver, interobserver, and interpullback variability for both lumen area and lesion length measurements in humans. Thus, FD-OCT can be proposed for precise analysis in the catheterization laboratory to guide decision making and in clinical trials focusing on imaging end points.