In sports sciences, blood lactate is commonly measured for performance testing or setting exercise intensity. Differences in measuring lactate concentration over time and across sampling sites during constant exercise are unknown. We aimed to compare blood lactate concentration across sampling sites during constant exercise. Eight participants performed three bouts of 20-min steady-state cycle exercise, consisting of 45, 60, and 75% of peak oxygen uptake (V ． O 2peak). Blood lactate levels were measured simultaneously from the fingertip and earlobe every 5 min during exercise. The time course analysis revealed that lactate level in the fingertip was significantly higher than in the earlobe at either work intensity (P < 0.05). A significant interaction effect (site x time) was observed at 60% V ． O 2peak (P < 0.001), which indicated that the difference in samples across sampling sites decreased over time. The levels in the fingertip samples were higher at 5 min (P < 0.05); this difference was not noted after 10 min. Similarly, the fingertip values were significantly higher until 10 min (P < 0.05), but not after 15 min at 75% V ． O 2peak. These findings suggest that the sampling site may affect the time course of change in lactate value under a constant workload.