Quantitative analysis of electrocardiographic ST-segment/heart rate relationship (ST/HR loop) during early recovery phase of exercise stress test provides a sensitive tool for the detection of coronary artery disease (CAD). This study evaluates the effect of data sampling frequency on the diagnostic performance of ST/HR loop in 1876 patients undergoing a routine exercise test on a bicycle ergometer. CAD was verified angiographically in 668 patients and excluded by coronary angiography (n = 119), myocardial scintigraphy (n = 250) and on clinical grounds (n = 839) in 1208 patients. The normalized ST/HR loop area was calculated in all cases by integration of ST-segment amplitude difference from the end of exercise to the end of the first 3 min of recovery period over HR and dividing the integral by the HR difference over the integration period. The effect of different sampling rates (one, two and five samples per minute) on the CAD discrimination ability of ST/HR loop area was subsequently evaluated using receiver operating characteristic (ROC) curves. Reduction in ST/HR data sampling frequency from two to one sample per minute resulted in a significantly decreased diagnostic performance of the ST/HR loop whereas no differences in CAD discrimination capacity were observed between sampling frequencies of two and five samples per minute. The choice of ST/HR data sampling frequency may have a significant impact on the CAD diagnostic ability of the ST/HR loop. The use of sampling frequency below two samples per minute results in a significantly diminished diagnostic performance, a fact that should be taken into consideration when employing ST/HR diagnostic procedures.