Purpose: Assessment of limitations in the perfusion dynamics of skeletal muscle may provide insight in the pathophysiology of exercise intolerance in, e.g., heart failure patients. Power doppler ultrasound (PDUS) has been recognized as a sensitive tool for the detection of muscle blood flow. In this volunteer study (N = 30), a method is demonstrated for perfusion measurements in the vastus lateralis muscle, with PDUS, during standardized cycling exercise protocols, and the test–retest reliability has been investigated. Methods: Fixation of the ultrasound probe on the upper leg allowed for continuous PDUS measurements. Cycling exercise protocols included a submaximal and an incremental exercise to maximal power. The relative perfused area (RPA) was determined as a measure of perfusion. Absolute and relative reliability of RPA amplitude and kinetic parameters during exercise (onset, slope, maximum value) and recovery (overshoot, decay time constants) were investigated. Results: A RPA increase during exercise followed by a signal recovery was measured in all volunteers. Amplitudes and kinetic parameters during exercise and recovery showed poor to good relative reliability (ICC ranging from 0.2–0.8), and poor to moderate absolute reliability (coefficient of variation (CV) range 18–60%). Conclusions: A method has been demonstrated which allows for continuous (Power Doppler) ultrasonography and assessment of perfusion dynamics in skeletal muscle during exercise. The reliability of the RPA amplitudes and kinetics ranges from poor to good, while the reliability of the RPA increase in submaximal cycling (ICC = 0.8, CV = 18%) is promising for non-invasive clinical assessment of the muscle perfusion response to daily exercise.