Objectives Measurement of blood glucose is essential for better control of diabetes mellitus. The pain associated with repeated blood sampling is a significant problem, and a less painful technique would be advantageous. This study was conducted to establish a less painful method of blood sampling for monitoring of blood glucose concentrations.Methods Fourteen healthy doctors and nurses at the age of 22 to 32 years were enrolled into this study. The earlobe was divided into 20 areas. Each area was punctured with a fine needle and the pain was assessed by the visual analog scale (VAS). Then, localized cooling or warming was applied before puncture and the pain was assessed by the VAS.Results The VAS tended to be lower toward the lower part of the earlobe. The VAS in the uppermost area was 53.1±19.1 mm and 28.9±16.8 mm in the lowest part of the earlobe. The VAS was higher in the uppermost area than in the lowest area in six of the seven volunteers (p=0.028). We did not find significant differences in the VAS and blood glucose levels between the control sampling and the warming, or cooling sampling. The localized warming shortens the time to obtain blood by 4.4 seconds (p= 0.0426).Conclusion The area of the ear, but not localized cooling or warming, significantly affects the pain in sampling blood from the earlobe. Blood sampling for glucose tests are recommended to be obtained at the lower pole of the earlobe.