Background: The etiology of sudden cardiac death in patients with end-stage renal disease (ESRD) on hemodialysis (HD) is largely unknown, though there is evidence to suggest that metabolic alkalosis induced by HD with a high-bicarbonate dialysate/prescription may play a role. Methods: We investigated the effects of metabolic alkalosis induced by HD with an acetate-containing bicarbonate-buffered dialysate on frequency of ventricular arrhythmia in 47 patients with ESRD on chronic HD using 48-h Holter monitoring in 3 phases: intra-HD, post-HD day 1, and post-HD day 2. Serum levels of bicarbonate, calcium, and potassium along with hemodynamics were measured pre-HD, post-HD, 20-h post-HD, and 44-h post-HD. Correlations were performed to verify the association between bicarbonate prescription and change in serum bicarbonate levels post-HD and to determine if the HD-induced change in serum bicarbonate level (metabolic alkalosis) had any direct association with ambient ventricular arrhythmia (premature ventricular contractions per hour) or indirect associations with ambient ventricular arrhythmia by affecting electrolytes or hemodynamics that are known to increase the risk of ventricular arrhythmia. Results: Mean pre-HD serum bicarbonate level was 21.3 mEq/L. Dialysate bicarbonate prescription (mean of 36.4 mEq/L) correlated with changes in serum bicarbonate levels immediately post-HD 26.7 mEq/L (r = 0.46, p < 0.01), 20-h post-HD 25.2 mEq/L (r = 0.38), and 44-h post-HD 23.2 mEq/L (r = 0.35, p = 0.01). No statistically significant correlations were found between the post-HD change in serum bicarbonate levels (metabolic alkalosis) with ambient ventricular arrhythmia, changes in serum calcium, potassium, or hemodynamics in any phase. Conclusions: High-bicarbonate dialysate prescription is associated with metabolic alkalosis following the HD procedure. A mild metabolic alkalosis induced by HD with an acetate-containing bicarbonate-buffered dialysate solution had no direct association with ambient ventricular arrhythmia on Holter monitoring and was not associated with changes in hemodynamics or changes in serum total calcium or potassium levels. This study helps to provide guidance for the safe use of high bicarbonate dialysate/prescription in patients with ESRD on HD.