The aim of this study was to determine blood pressure (BP) and heart rate (HR) responses triggered during an isokinetic testing protocol in professional soccer players and compare cardiovascular parameters at completion of this isokinetic protocol with those during a treadmill test. Using purposive sampling, 63 professional soccer players were recruited. Cardiovascular responses were measured noninvasively during a bilateral testing protocol of knee flexion and extension. Treadmill ergospirometry following an incremental speed protocol was performed to analyze the same cardiovascular parameters at rest and at completion of this test. There were significant differences in diastolic blood pressure (DBP) and HR according to field position. The parameters presented high homogeneity at both competitive levels. Systolic blood pressure, mean arterial pressure, HR, and rate pressure product at completion of the treadmill test were significantly higher than those at completion of the isokinetic protocol. Intermittent isokinetic testing protocol of the knee triggers normal and safe BP and HR responses in healthy professional soccer players. The HR of the defenders was higher than those of the forwards and midfielders but was independent of the competitive level. The values of cardiovascular parameters at isokinetic protocol completion were lower than those during the treadmill test.