In recent years, the number of hemodialysis patients is increasing due to population aging and lifestyle-related diseases, and hemodialysis lowers patients’ quality of life. The technique of hemodialysis was developed based on hemolysis, and it is thought that hemolysis in hemodialysis is induced during discharge by the peristaltic pump. However, studies have not investigated in detail whether hemolysis is instantaneously induced either by the pump or the accumulation of mechanical damage to erythrocytes. In this study, the deformability and degree of hemolysis of erythrocytes in extracorporeal circulation during hemodialysis were measured quantitatively, based on the mechanics of an in vitro model experiment using commercially available blood. In this experiment, the influence of drug administration to patients could be ignored. In the experiment, blood was circulated in a hemodialysis circuit model in which the parameters of flow rate and circulation time were set with reference to hemodialysis conditions in clinical settings. Blood for measurement was sampled from the circuit. The deformability of the sampled erythrocytes was evaluated based on the time constant of shape recovery, which was measured using two methods;the micro-channel and uni-axial stretching methods. Hemolysis was quantified based on absorbance measured using a spectrometer. The results showed that the deformability of erythrocytes remained nearly unchanged over time of hemodialysis when measured by both methods, whereas hemolysis increased in all conditions. These findings indicate that hemolysis is induced instantaneously by the peristaltic pump in the hemodialysis system.