Effects of online hemodiafiltration (HDF) using acetate-free bicarbonate dialysis (AFD) fluid on microinflammation, resulting in improved nutritional status in hemodialysis patients, were examined and compared with conventional acetate-containing bicarbonate dialysis (ACD) fluid. A total of 24 hemodialysis patients were registered for a cross-over design study for a 6-month period. These patients were subjected to ACD for the first 3 months followed by AFD fluid for the latter 3 months. Blood variables of C-reactive protein (CRP), interleukin-6 (IL-6), leptin, neuropeptide Y (NPY), protein catabolic rate (PCR) and %creatinine (Cr) index were determined after the first and last 3-month period. The filters and the conditions of HDF and drug regimens including erythropoiesis-stimulating agents were unchanged throughout the cross-over study. Predialysis blood pH and bicarbonate were significantly higher in the AFD phase than in the ACD phase. Blood CRP and IL-6 levels were significantly decreased in the AFD group compared to the ACD group. Concerning nutritional evaluation, leptin and NPY were significantly lower and higher, respectively, in the AFD phase than in the ACD phase. PCR tended to be higher in the AFD phase than in the ACD phase. A significantly higher %Cr index level was observed in the AFD phase than in the ACD phase. These results suggest that online HDF using AFD fluid contributes to alleviating bioincompatible events associated with microinflammation, leading to improvement in the nutritional status in hemodialysis patients.