PURPOSE: Hemoglobin mass (Hbmass) is commonly assessed using the CO re-breathing method with the subject in the seated position. This may lead to an underestimation of Hbmass as blood in lower extremity veins while seated may not be tagged with carbon monoxide (CO) during the re-breathing period. METHODS: To test this hypothesis, CO re-breathing was performed on four occasions in nine male subjects, twice in the seated position and twice in combination with light cycle ergometer exercise (1 W/kg body-weight) intending to accelerate blood circulation and thereby potentially allowing for a better distribution of CO throughout the circulation as compared to in the seated position. Blood samples were drawn from an antecubital vein and the saphenous magna vein following the re-breathing procedure. RESULTS: In the seated position, CO re-breathing increased the percent carboxyhemoglobin (%HbCO) in the antecubital vein to 8.9 % (7.8-10.7) [median (min-max)], but less (P = 0.017) in the saphenous magna vein [7.8 % (5.0-9.9)]. With exercise, no differences in %HbCO were observed between sampling sites. As a result, CO re-breathing in combination with exercise revealed a ~3 % higher (P = 0.008) Hbmass, i.e., 936 g (757-1,018) as compared to 908 g (718-940) at seated rest. CONCLUSION: This study suggests an uneven distribution of CO in the circulation if the CO re-breathing procedure is performed at rest in the seated position and therefore can underestimate Hbmass.