The measurement of relative blood volume (RBV) changes during ultrafiltration assume a constant mass and distribution of circulating blood components such as hematocrit. The authors examine the validity of this assumption in 10 subjects undergoing repeated direct measurements of systemic hematocrit and plasma volume (PV(icg)) using indocyanine green dilution at four stages of dialysis with intermittent ultrafiltration. Ultrasonic RBV changes were monitored. Absolute blood volumes (ABV) were initially derived for each PV(icg) estimate, and corresponding measured systemic hematocrit was adjusted by a factor of 0.86 to correct for the difference between the systemic and whole-body hematocrit (constant Fcell ratio). PV(icg) and ABV changes correlated closely (R = 0.98; P <0.001). ABV changes overestimated reduction in PV(icg) during ultrafiltration (mean difference, -140 +/- 202 ml). The calculated red cell mass however was variable (P <0.01). Fcell ratio was then adjusted at each blood volume measurement (Fcell(1), 0.87 +/- 0.02; Fcell(2), 0.89 +/- 0.03; Fcell(3), 0.94 +/- 0.06; Fcell(4), 0.94 +/- 0.04; P <0.01) to maintain a constant red cell mass (2146 +/- 460 ml). When ABV was recalculated using PV(icg), systemic hematocrit and variable Fcell (ABV(Fvariable)), the mean difference between PV(icg) changes and ABV(Fvariable) changes, was negligible (-0.2 +/- 35 ml). During intermittent ultrafiltration, RBV changes systematically underestimated the percentage reduction in ABV (mean difference, 7.7 +/- 10.6%). When corrected for variations in Fcell, ABV(Fvariable) and RBV differences were negligible (mean difference 1.2 +/- 2.6%). Varying Fcell ratio probably reflects microvascular volume change with net fluid shift from the microcirculation to macrocirculation (intravascular refill). This may result in underestimation of changes in systemic hematocrit and RBV during dialysis such that they were less than those predicted by directly measured changes in plasma volume.