Background/Aims: Previous studies have suggested that online hemodiafiltration (OL-HDF) fluid can be used as dialysate for continuous renal replacement therapies, and thus HDF costs can be reduced. The aims of this study were to determine the purity of OL-HDF fluid and to verify the stability of the electrolyte composition and acid-base balance during its storage. Methods: OL-HDF fluid was collected in 70 individual bags and stored for up to 7 days. The following tests were performed daily in 10 bags: natural visible precipitation ( macrocrystallization), sample collection for chemical analysis and fluid culture, limulus amebocyte lysate endotoxin test, standard culture of NALGENE® filters after passing of the fluid, and molecular analysis of bacterial DNA. Results: The values of pH and pCO<sub>2</sub> showed a significant change starting at 24 h (p < 0.001); after 72 h, their values were beyond the measurable range. Coefficient of variation for pCO<sub>2</sub> was as high as 25.7%. Electrolyte composition (Na<sup>+</sup>, K<sup>+</sup>, Cl<sup>–</sup>, Ca<sup>2+</sup> and glucose) showed a statistically significant difference over time (p < 0.05); however, their coefficients of variation were low (1.7, 1.4, 0.6, 2.3 and 0.9%, respectively), which might not be considered clinically significant. Negative results were obtained at all points by fluid and filter cultures, endotoxin test and molecular analysis. No macrocrystallization was observed at any time point. Conclusions: We demonstrate the microbiological purity of OL-HDF fluid stored for up to 7 days. The electrolyte composition was stable, except for a relevant change in p<smlcap>CO</smlcap><sub>2</sub> and consequently in pH (first noted at 24 h), emphasizing the need to reassess the acid-base balance in multilayer plastic bags in future studies.