Objectives: The microbiological safety must be ensured because a large amount of substitution fluid is directly injected into the blood stream of the patients during online hemodiafiltration (HDF). When using a central dialysis fluid delivery system (CDDS) for online HDF, the microbiological safety becomes even more important because of the structural risk of microbiological contamination. This study aims at determining the microbiological safety of substitution fluid in high-volume online HDF using CDDS. Methods: All samples collected by aseptic technique during high-volume HDF. Sample collection was performed at three sites (Fig-1): (1) a port on the pre-CDDS, (2) a port on the post-submicrofilter of CDDS, and (3) a port on the machine post-dialysate ultrafilter (1st & 2nd endotoxin retentive filter) fluid. Substitution fluid was sampled from all HDF machines (27 machines) performing HDF. While the online HDF was running, 100 mL of the substitution fluid was collected using a sterile sampling kit. Bacterial culture and LAL (limulus amebocyte lysate) test for bacterial endotoxin detection were performed by the standard methods. Results: Microbiology count results showed <0.1 CFU/mL in all substitution fluid samples collected from 27 HDF machines. Endotoxin concentration was <0.005 EU/mL in all substitution fluid samples. Samples obtained from a port of pre-CDDS and post-submicrofilter of CDDS showed microbiology count of <0.1 CFU/mL and endotoxin concentration of <0.01 EU/mL.  Conclusions: These results show the microbiologic safety of substitution fluid produced by CDDS for high-volume HDF.