Background: The aim of the present study was to assess hemoglobin changes occurring at the beginning of high-flux hemodialysis (HD). Methods: In a group of 20 chronic HD patients (group A), total hemoglobin (tHb), hematocrit (Hct) and total serum proteins (TP) were measured in blood samples drawn from an arterial fistula needle before the initiation of high-flux HD, and from an arterial line 5 min after HD with the dialysate in the bypass mode. 31 chronic stable HD patients (group B) served as controls. In group B patients, tHb was measured in blood samples drawn from an arterial fistula needle before the initiation of high-flux HD, and from arterial and venous lines simultaneously 5 min later. Blood flow rates in groups A and B were set from the beginning of the study to 300 ml/min, while the bicarbonate dialysate flow rate and ultrafiltration rate in group B patients was set to 700 ml/min and zero, respectively. The same high-flux dialyzer was used for all patients (FLX-18, membrane PEPA 1.8 m<sup>2</sup>). Results: A comparison of baseline (pre-dialysis) values with those derived from an analysis of the arterial line in groups A and B at 5 min revealed that tHb decreased by 0.6 ± 0.2 g/dl (5.2 ± 1.7%, p < 0.001) and 0.7 ± 0.7 g/dl (5.4 ± 6.2%, p < 0.001), respectively. At the same time, Hct and TP in group A decreased by 1.32 ± 0.7% (3.8 ± 2.0%, p <0.001) and 0.3 ± 0.1 g/dl (4.8 ± 1.4%, p < 0.001), respectively. Blood volume (BV) and plasma volume (PV) in group A patients at 5 min as calculated from tHb and TP values increased by 5.6 ± 1.9 and 5.2 ± 1.7%, respectively, while BV in group B patients increased by 6.1 ± 7.0% (not significant when compared to group A). tHb did not change significantly in 14 patients (group C) studied immediately after adopting the supine position and 5 min later in the absence of HD. Conclusion: A 5% decrease in tHb was observed 5 min after the initiation of high-flux HD with a zero ultrafiltration rate, and was due to an increase in BV.