Purpose: Hemoglobin mass (Hb_mass) is commonly assessed using the CO re-breathing method with the subject in the seated position. This may lead to an underestimation of Hb_mass 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) Hb_mass, 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 Hb_mass.