Objectives: To evaluate whether the donation of 900 mL of blood reduces the central blood volume (CBV) assessed by thoracic electrical impedance (TI) and plasma pro-atrial natriuretic peptide (proANP). Background: Donation of 450 mL of blood carries a 1% risk of a vasovagal reaction. Withdrawal of 900 mL of blood decreases cardiac output; however, the effect on CBV remains unknown. Methods/Materials: A randomised, single-blinded, placebo-controlled, crossover design was used, where 21 healthy semi-recumbent men donated 2 × 450 mL blood or were sham-phlebotomised. Changes in CBV were estimated by proANP and TI at 1.5 (TI 1.5) and 100 (TI 100) kHz, reflecting extracellular volume and (regional) total body water, respectively, and the index value (IDX; 1/T 1.5-1/TI 100) was used to estimate changes in intracellular (red cell) volume. Systolic, diastolic and mean arterial blood pressure; heart rate; stroke volume; cardiac output; and systemic vascular resistance were monitored. After completion of the study, 1000 mL of isotonic saline was infused. Results: Changes (mean% ± SD) in TI 1.5 , TI 100 and IDX were similar after 450 mL (−0.2 ± 1.6%, 0.0 ± 1.1%, −0.4 ± 10.1%) and 900 mL (0.1 ± 1.6%, 0.2 ± 1.5% and −2.0 ± 15.8%) of blood donation compared to after a sham donation of 450 mL (−0.9 ± 1.2%, −0.5 ± 1.5% and −0.1 ± 6.1%) and 900 mL (−1.2 ± 1.5%, −0.6 ± 1.3% and 0.5 ± 9.9%). In addition, changes in plasma proANP were similar after 450 and 900 mL of blood donation (−0.8 ± 6.7% and −7.6 ± 7.9%) as after sham donations (1.3 ± 7.3% and −4.5 ± 5.6%). Monitoring haemodynamic variables revealed that stroke volume decreased after the donation of 900 mL of blood (−12 ± 12 mL) compared to sham donations. Conclusion: During a 900-mL blood loss in semi-recumbent men, CBV measured by TI and plasma proANP is not affected.