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Introduction: We aimed to compare prospectively the effect of high‐flux hemodialysis and post‐dilution hemodiafiltration on platelets. Methods: Twenty‐two hemodialysis patients were treated with one high‐flux hemodialysis and one post‐dilution hemodiafiltration procedure. PFA‐100 closure times (collagen/epinephrine—CEPI and collagen/adenosine diphosphate—CADP) were measured before and after the procedure, as well as platelet count, hemoglobin, hematocrit, and red blood cell count. All pre‐dialysis and post‐dialysis samples were taken from the afferent line. Findings: The platelet count after vs. before hemodialysis did not change significantly (229.3 ± 55.0 x109/L vs. 233.6 ± 55.8 × 109/L; P = 0.269), but was significantly lower after post‐dilution hemodiafiltration (215.5 ± 51.7 × 109/L vs. 245.3 ± 59.9 × 109/L; P < 0.0001). CEPI after vs. before hemodialysis was not significantly prolonged (192.9 ± 60.8 s vs. 173.4 ± 52.5 s; P = 0.147), and the same applied to CADP (143.6 ± 40.3 s vs. 142.6 ± 38.4 s; P = 0.897). CEPI after vs. before post‐dilution hemodiafiltration was significantly prolonged (268.3 ± 41.3 s vs. 176.4 ± 54.0 s; P < 0.0001) as was CADP (221.0 ± 53.9 s vs.133.9 ± 31.1 s; P < 0.0001). Discussion: Only after post‐dilution hemodiafiltration, we found a lower platelet count and prolonged platelet closure times. [ABSTRACT FROM AUTHOR]