Total hemoglobin mass (Hb_mass) 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_0.5) compared to 1.5 mL/kg (CO_1.5) 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_0.5 (CO_0.5-1 and CO_0.5-2) or CO_1.5 (CO_1.5-1 and CO_1.5-2) was administered. Venous blood samples to determine %HbCO and quantify Hb_mass were obtained prior to, and at 6 (T₆), 8 (T₈) and 10 min (T₁₀) 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_mass between CO_0.5-1 and CO_0.5-2 was 26 g (–26; 79 g) and between CO_1.5-1 and CO_1.5-2, it was 17 g (–18; 52 g)_. The respective typical error (TE) corresponded to 2.4% (CO_0.5) and 1.5% (CO_1.5), while it was 6.5% and 3.0% at 2320 m. With CO_0.5, shortening the CO rebreathing resulted in a TE for Hb_mass of 4.4% (T₈ vs. T₁₀) and 14.1% (T₆ vs T₁₀) and with CO_1.5, 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%.