Hemodialysis is accompanied by osmotic perturbations with distinct effects on red blood cell, plasma, and blood volumes. A series of in vitro studies was done to analyze the separate effect on cell volume. Whole porcine blood was circulated through an extracorporeal circulation maintaining a constant blood volume. Hemoconcentration was continuously measured by established optical and ultrasonic online techniques. Osmotic perturbation was performed by variation of dialysate conductivity within the clinical range of 13-15 mS/cm. Blood samples were analyzed using a microcentrifuge and a standard cell counter. As dialysate conductivity increased, centrifuge hematocrit (in %) decreased with a slope of -1.91% per unit of conductivity in mS/cm (r2 = 0.98). At the same time, Coulter-Counter hematocrit slightly decreased only by -0.18% (r2 = 0.53), while optical and ultrasonic hematocrit showed a small increase by 0.44% (r2 = 0.97) and 0.69% (r2 = 0.94) per unit of conductivity in mS/cm. The sensitivity to osmotic perturbation is consistent with theory and with specific characteristics of measuring techniques used in this study. The differences, however, need to be considered when comparing measurements obtained by different techniques. Finally, devices used for relative blood volume measurement in hemodialysis should be insensitive to osmosis-induced changes in red blood cell volume.