Objective: The purpose of this study was to evaluate the effects of electrical muscle stimulation (EMS) on muscles, using 99mTc-sestamibi SPECT/CT. Materials and Methods: We prospectively enrolled 20 consecutive male professional water polo players. The mean age was 25 years (range, 18-36 years). All athletes underwent 99mTc-sestamibi SPECT/CT of the thigh (rectus femoris and vastus medialis muscle groups) before and after EMS. Images were quantified to identify increases in perfusion after EMS. Results: Before EMS, there were no significant differences between the right and left thigh (rectus femoris and vastus medialis muscles) in terms of perfusion (p = 0.4). However, the comparison between the pre- and post-EMS analyses of the same muscle groups showed significant differences in radiotracer uptake (p < 0.001), with a mean increase in perfusion of 128% for the rectus femoris muscle group (95% CI: 0.86-1.61) and 118% for the vastus medialis muscle group (95% CI: 0.96-1.79). Conclusion: 99mTc-sestamibi SPECT/CT is an objective means of evaluating blood flow in muscles submitted to EMS, which appears to promote significant increases in such blood flow.