Background: The analysis of heart rate variability (HRV) is a useful tool to evaluate cardiac autonomic modulation, which is frequently impaired in chronic uremia. Aims: The aim of this study was to evaluate HRV in chronic uremics and to separately investigate the acute changes induced by volume depletion and solute removal during a hemodialysis session. Methods: Fourteen uremic patients (8 males and 6 females, aged 50 ± 15 years) on maintenance hemodialysis and 14 sex- and age-matched healthy controls were studied. Both groups underwent ambulatory electrocardiogram monitoring to evaluate the HRV time and frequency domain indices. The hemodialysis session was performed by 1 h of high-rate isolated ultrafiltration followed by 3 h of bicarbonate diffusive procedure. Results: In uremic patients, the overall variability in the frequency [low-frequency power (LF): 505 ± 473, vs. 1,446 ± 654; high-frequency power (HF): 133 ± 162 vs. 512 ± 417; p < 0.001] and time domain indices (standard deviation of normal R-R intervals: 101.9 ± 33.3 vs. 181.7 ± 44.1 ms; p < 0.001) was markedly reduced compared to controls, whereas mean heart rate (83 ± 12.4 vs. 60.9 ± 8.8 bpm; p < 0.001) and LF/HF ratio (5.8 ± 3.5 vs. 2.2 ± 0.8; p < 0.001) were increased. Isolated ultrafiltration produced a marked further decrease in HRV indices, but the subsequent diffusive hemodialysis procedure, with a low ultrafiltration rate, made HRV increase again. Conclusions: Chronic uremics showed abnormal autonomic modulation with sympathetic-vagal imbalance. The unbalanced hypersympathetic response to body fluid depletion is related to the ultrafiltration rate. Low interdialytic weight gain and a low ultrafiltration rate, associated with adequate hemodialysis, should be the preferable strategy for uremic patients with autonomic dysfunction.