We examined whether a semi-automated carbon monoxide (CO) rebreathing method accurately detect changes in blood volume (BV) and total hemoglobin mass (tHb). Furthermore, we investigated whether a supine position with legs raised reduced systemic CO dilution time, potentially allowing a shorter rebreathing period. Nineteen young healthy males participated. BV and tHb was quantified by a 10-min CO-rebreathing period in a supine position with legs raised before and immediately after a 900 ml phlebotomy and before and after a 900 ml autologous blood reinfusion on the same day in 16 subjects. During the first CO-rebreathing, arterial and venous blood samples were drawn every 2 min during the procedure to determine systemic CO equilibrium in all subjects. Phlebotomy decreased (P < 0.001) tHb and BV by 166 ± 24 g and 931 ± 247 ml, respectively, while reinfusion increased (P < 0.001) tHb and BV by 143 ± 21 g and 862 ± 250 ml compared to before reinfusion. After reinfusion BV did not differ from baseline levels while tHb was decreased (P < 0.001) by 36 ± 21 g. Complete CO mixing was achieved within 6 min in venous and arterial blood, respectively, when compared to the 10-min sample. On an individual level, the relative accuracy after donation for tHb and BV was 102-169% and 55-165%, respectively. The applied CO-rebreathing procedure precisely detect acute BV changes with a clinically insignificant margin of error. The 10-min CO-procedure may be reduced to 6 min with no clinical effects on BV and tHb calculation. Notwithstanding, individual differences may be of concern and should be investigated further.