The purpose of this study was to explore the feasibility of performing rest and exercise technetium-99m-sestamibi (99mTc-sestamibi) studies on the same day. We prospectively studied 34 asymptomatic volunteers using two different protocols (17 patients studied using one-day protocol and 17 patients studied using two-day protocol). For the one-day protocol, the rest study was performed first. For the two-day protocol, the exercise study was performed first followed by a second-day rest study. Left and right ventricular ejection fractions, myocardium/lung, myocardium/liver, myocardium/spleen, and myocardium/gallbladder count ratios were similar for the one- and two-day groups. No significant difference was observed in any variable measured. These data suggest that 99mTc-sestamibi can be used for rest and treadmill exercise studies performed on the same day to provide assessment of ventricular function and perfusion similar to studies performed on separate days.