Online hemodiafiltration (OL-HDF) is a blood purification therapy based on diffusion and ultrafiltration and is classified into two types according to the mode of addition of the substitution fluid: pre-dilution OL-HDF (pre-HDF) and post-dilution OL-HDF (post-HDF); we previously reported that pre-HDF is more biocompatible. However, we used lower blood and substitution flow rates in that study and may not have accurately simulated the treatment conditions used in Europe. In this study, we compared the biocompatibilities of the treatment conditions of pre-HDF, commonly used in Japan, and post-HDF, commonly used in Europe, to determine the most biocompatible treatment conditions. We compared the biocompatibilities of pre-HDF and post-HDF using high blood flow rates and high substitution fluid volumes, and also compared the results with those of our previous study. We enrolled six stable patients undergoing maintenance dialysis at our clinic for this study. After the patients underwent hemodialysis (HD), post-HDF, and pre-HDF treatment, the biocompatibilities (based on the serum levels of high-sensitivity C-reactive protein, interleukin-6, pentraxin-3, β-thromboglobulin, and soluble P-selectin, and the results of the lymphocyte blastogenesis test using phytohemagglutinin and concanavalin A as mitogens) and removal performances (removal performance for urea, creatinine, β2-microglubulin [MG], and α1-MG, and albumin leakage) were determined. There were no significant differences in the biocompatibility parameters evaluated among the three treatment modes. Post-HDF was associated with significantly higher removal rates of β2-MG than HD. Post-HDF was associated with significantly higher removal rate of α1-MG, and also significantly higher albumin leakage, than HD and pre-HDF.