Objective: We attempt to identify heart rate variability (HRV) components and decide whether or not such components are more sensitive to the hemodialysis (HD) process by excluding the presence of comorbid conditions known to affect HRV. Methods: It was a prospective cohort study of factors associated with HRV. Thirty-five HD patients were admitted to the study. The research was divided into three phases. During the first phase, baseline Holter and blood pressure recordings were carried out. Then in the second phase, the patients were admitted for HD with continuous Holter and blood pressure recording. The third phase was after HD with continuous Holter and blood pressure recording. The frequency and time domain analyses of HRV were carried out by an independent, blinded investigator. Plasma Na<sup>+</sup>, K<sup>+</sup>, ionized Ca, hemoglobin, serum creatinine and blood urea nitrogen (BUN) were checked 1 h before and 2 h after HD. Results: After HD, the serum creatinine and BUN were obviously reduced (p < 0.01), which was also expressed by fractional urea clearance (K<sub>t</sub>/V; 1.55 ± 0.25), which means efficient HD. Standard deviation of all RR intervals and low-frequency/high-frequency (LF/HF) ratio were reduced significantly (p < 0.05) at the ‘HD for 3 h’ time point while the blood pressure levels were relatively stable during the HD process. The ultrafiltration rate and K<sub>t</sub>/V appeared to be the main determinants of the LF/HF ratio in HD. In our cases, the LF/HF ratio positively correlated with K<sub>t</sub>/V and negatively correlated with ultrafiltration volume. Conclusion: Our study suggested a shift in sympathovagal balance toward sympathetic activation during the HD process. Sympathetic overactivity is positively correlated with the ultrafiltration rate, and negatively correlated with the K<sub>t</sub>/V which means that the HD process itself affects HRV parameters indeed.