The circadian rhythm of heart rate is well known. The purpose of this study was to investigate the characteristics of the relation between heart rate and oxygen uptake during a graded exercise testing with and without previous exercise. Eight healthy male students, aged 18 to 21 years, performed the graded sub-maximal exercise two times using a treadmill, after walking on a treadmill at a speed of 60 m/min for a total of 4 hours (2 hours in the morning, 2 hours in the afternoon) and without previous prolonged walking. The apparent resting heart rate after one hour of walking in the afternoon (82.3±9.1 beats mm) was higher than in the morning (71.3±5.8 beats/min), and this phenomenon was observed continuously exercise heart rate at the end of 2 hours of walking (afternoon : 96.4±5.4, morning: 87.2±7.3 beats min) . On the other hand, the oxygen uptake during exercise did not alter at the end of walking (afternoon: 622±85, morning: 600±133 ml/min) . This walking intensity ranged from 17.1 to 26.6, with a mean of 20.5 percent of maximal oxygen uptake. The pre-exercise resting heart rate before the exercise testing altered from -4.8 to 13.1 beats/min with and without the presence of walking. The observed individual regression line between heart rate and oxygen uptake from the testing shifted along with the altered resting heart rate. Therefore, the calculated individual new regression lines with and without walking between net heart rate, excluding pre-exercise rest, and oxygen uptake were approximate to a constant line. It was concluded that the exercise energy expenditure could be calculated accurately (-2 to 7 % of errors) using net heart rate, as compared with overestimations of the actual energy cost using the higher resting heart rate.