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Objectives Positron emission tomography (PET) quantifies stress myocardial perfusion (in cc/min/g) and coronary flow reserve to guide noninvasively the management of coronary artery disease. This study determined their test-retest precision within minutes and daily biological variability essential for bounding clinical decision-making or risk stratification based on low flow ischemic thresholds or follow-up changes. Background Randomized trials of fractional flow reserve–guided percutaneous coronary interventions established an objective, quantitative, outcomes-driven standard of physiological stenosis severity. However, pressure-derived fractional flow reserve requires invasive coronary angiogram and was originally validated by comparison to noninvasive PET. Methods The time course and test-retest precision of serial quantitative rest-rest and stress-stress global myocardial perfusion by PET within minutes and days apart in the same patient were compared in 120 volunteers undergoing serial 708 quantitative PET perfusion scans using rubidium 82 (Rb-82) and dipyridamole stress with a 2-dimensional PET-computed tomography scanner (GE DST 16) and University of Texas HeartSee software with our validated perfusion model. Results Test-retest methodological precision (coefficient of variance) for serial quantitative global myocardial perfusion minutes apart is ±10% (mean ΔSD at rest ±0.09, at stress ±0.23 cc/min/g) and for days apart is ±21% (mean ΔSD at rest ±0.2, at stress ±0.46 cc/min/g) reflecting added biological variability. Global myocardial perfusion at 8 min after 4-min dipyridamole infusion is 10% higher than at standard 4 min after dipyridamole. Conclusions Test-retest methodological precision of global PET myocardial perfusion by serial rest or stress PET minutes apart is ±10%. Day-to-different-day biological plus methodological variability is ±21%, thereby establishing boundaries of variability on physiological severity to guide or follow coronary artery disease management. Maximum stress increases perfusion and coronary flow reserve, thereby reducing potentially falsely low values mimicking ischemia. [ABSTRACT FROM AUTHOR]