The non-invasively measured initial systolic time interval (ISTI) reflects a time difference between the electrical and pumping activity of the heart and depends on cardiac preload, afterload, autonomic nervous control and training level. However, the duration of the ISTI has not yet been compared to other time markers of the heart cycle. The present study gauges the duration of the ISTI by comparing the end point of this interval, the C-point, with heart cycle markers obtained by echocardiography. The heart rate of 16 healthy subjects was varied by means of an exercise stimulus. It was found that the C-point, and therefore the end point of ISTI, occurred around the moment of the maximum diameter of the aortic arch in all subjects and at all heart rates. However, while the time difference between the opening of the aortic valves and the maximum diameter of the aortic arch decreased significantly with decreasing RR-interval, the time difference with respect to the moment of the C-point remained constant within the subjects. This means that the shortening of the ISTI with increasing heart rate in response to an exercise stimulus was caused by a shortening of the pre-ejection period (PEP). It is concluded that the ISTI can be used as a non-invasive parameter indicating the time difference between the electrical and mechanical pumping activity of the heart, both inside and outside the clinic.