During water immersion in humans, the use of changes in hematocrit (Hct) and hemoglobin concentration (Hb) underestimates the relative changes in plasma volume (PV) as measured directly with Evans blue (EB). It is not known whether the same is the case during posture changes. Therefore, changes in PV were determined with an EB dilution technique in 10 males before, during, and after an acute posture change from seated to 6° head-down tilt (HDT). The EB method was improved to take into account changes in transcapillary escape rate of albumin-bound EB. Furthermore, blood was sampled from a central venous catheter. Hct and Hb were simultaneously measured. During HDT, PV determined with EB increased by 9.3 ± 2.0% but increased only 4.5 ± 0.9% when calculated with the Hct/Hb method (P < 0.05 vs. EB measurements). Thus use of the Hct/Hb method in humans leads to underestimation of the change in PV by as much as 50% during an acute change in posture. Therefore, a direct tracer-dilution method must be used for accurate estimations of changes in PV during changes in posture or other antiorthostatic maneuvers.