Total hemoglobin mass (Hb) is routinely assessed in studies by the carbon monoxide (CO) rebreathing. Its clinical application is often hindered due to the consequent rise in carboxyhemoglobin (%HbCO) and the concern of CO toxicity. We tested the reproducibility of the CO rebreathing with a CO dose of 0.5 mL/kg body mass (CO) compared to 1.5 mL/kg (CO) and when shortening the CO rebreathing protocol. Therefore, CO rebreathing was performed 1×/day in eight healthy individuals on four consecutive days. On each day, either CO (CO-1 and CO-2) or CO (CO-1 and CO-2) was administered. Venous blood samples to determine %HbCO and quantify Hb were obtained prior to, and at 6 (T), 8 (T) and 10 min (T) of CO rebreathing. This protocol was tested at sea level and at 2320 m to investigate the altitude-related measurement error. At sea level, the mean difference (95% limits of agreement) in Hb between CO-1 and CO-2 was 26 g (-26; 79 g) and between CO-1 and CO-2, it was 17 g (-18; 52 g) The respective typical error (TE) corresponded to 2.4% (CO) and 1.5% (CO), while it was 6.5% and 3.0% at 2320 m. With CO, shortening the CO rebreathing resulted in a TE for Hb of 4.4% (T vs. T) and 14.1% (T vs T) and with CO, TE was 1.6% and 5.8%. In conclusion, the CO dose and rebreathing time for the CO rebreathing procedure can be decreased at the cost of a measurement error ranging from 1.5-14.1%.