Anaemia is defined by the concentration of haemoglobin (Hb). However, this value is dependent upon both the total circulating haemoglobin mass (tHb-mass) and the plasma volume (PV)-neither of which is routinely measured. Carbon monoxide (CO)rebreathing methods have been successfully used to determine both PV and tHb-mass in various populations. However, these methods are not yet suitable for ventilated patients. This study aimed to modify the CO-rebreathing procedure such that a single inhalation of a CO bolus would enable its use in ventilated patients. Eleven healthy volunteers performed four CO-rebreathing tests in a randomized order, inhaling an identical CO volume. In two tests, CO was rebreathed for 2 min (optimized CO rebreathing; oCOR), and in the other two tests, a single inhalation of a CO bolus was conducted with a subsequent breath hold of 15 s (Proc new 15s) or 30 s (Proc new 30s). Subsequently, the CO volume in the exhaled air was continuously determined for 20 min. The amount of CO exhaled after 7 and 20 min was respectively 3.1 ± 0.3 and 5.9 ± 1.1 ml for oCOR, 8.7 ± 3.6 and 12.0 ± 4.4 ml for Proc new 15s and 5.1 ± 2.0 and 8.4 ±2.6 ml for Proc new 30s. tHb-mass was 843 ± 293 g determined by oCOR, 821 ± 288 g determined by Proc new 15s (difference: P < 0.05) and 849 ± 311 g determined by Proc new 30s. Bland-Altman plots demonstrated slightly lower tHb-mass values for Proc new 15s compared with oCOR (−21.8 ± 15.3 g) and similar values for Proc new 30s. In healthy volunteers, a single inhalation of a CO bolus, preferably followed by a 30 s breath hold, can be used to determine tHb-mass. These results must now be validated for ventilated patients. K E Y W O R D S blood volume, carboxy-haemoglobin, CO rebreathing, ventilated patients This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.