The aim of this study was to evaluate the reproducibility of myocardial blood flow (MBF) and myocardial flow reserve (MFR) measurement in patients referred for dynamic SPECT. Methods: We retrospectively analyzed patients referred for myocardial perfusion imaging. SPECT data were acquired on a cadmium zinc telluride-based pinhole cardiac camera in list mode using a stress (251 ± 15 MBq)/rest (512 ± 26 MBq) 1-d 99mTc-tetrofosmin protocol. Kinetic analyses were done with software using a 1-tissue-compartment model and converted to MBF using a previously determined extraction fraction correction. MFR was analyzed and compared globally and regionally. Motion detection was applied, but not attenuation correction. Results: In total, 124 patients (64 male, 60 female) were included, and SPECT acquisitions were twice reconstructed by the same nuclear medicine board-certified physician for 50 patients and by 2 different physicians for 74. Both intra- and interobserver measurements of global MFR had no significant bias (-0.01 [P = 0.94] and 0.01 [P = 0.67], respectively). However, rest MBF and stress MBF were significantly different in global left ventricular evaluation (P = 0.001 and P = 0.002, respectively) and in the anterior territory (P < 0.0001) on interuser analysis. The average coefficient of variation was 15%-30% of the mean stress MBF if the analysis was performed by the same physician or 2 different physicians and was around 20% of the mean MFR independently of the processing physician. Using the MFR threshold of 2, we noticed good intrauser agreement, whereas it was moderate when the users were different (κ = 0.75 [95% CI, 0.56-0.94] vs. 0.56 [95% CI, 0.36-0.75], respectively). Conclusion: Repeated measurements of global MFR by the same physician or 2 different physicians were similar, with an average coefficient of variation of 20%. Better reproducibility was achieved for intrauser MBF evaluation. Automation of processing is needed to improve reproducibility.