Background: According to several studies the QRS amplitude of the ECG increases during hemodialysis. The detailed background to this phenomenon has not been defined. Two main mechanisms have been suggested: myocardial ischemia and volume changes. New noninvasive technologies make possible a comparison of QRS complex changes synchronously with myocardial ischemia and extracellular water (ECW)/blood volume (BV) changes during hemodialysis. Methods: In this study hemodialysis-related changes in body weight, biochemical blood variables, BV, ECW, ST segment and QRS complex were analyzed in 15 patients (age 36–76, time on dialysis 0–6 years) undergoing chronic hemodialysis treatment. QRS complex and ST segment changes were measured using a dynamic vectorcardiographic monitoring system. The ECG parameters measured were QRS vector difference (QRS-VD) and ST vector magnitude (ST-VM6). Bioimpedance analysis was used to detect changes in the ECW. Continuous measurement of BV changes was implemented using an on-line optical reflection method based on the reflection of infrared light by erythrocyte membranes. Blood hemoglobin (B-Hb), hematocrit (B-Hcr), plasma sodium (P-Na), chloride (P-Cl), magnesium (P-Mg), potassium (P-K), ionized calcium (P-iCa), phosphate (P-Pi), creatinine (P-Crea) and urea (S-Urea) were monitored. Results: The mean QRS-VD increase during the dialysis session was almost fourfold (372 ± 300%) from 4.16 ± 2.40 to 15.60 ± 7.0 μVs (p < 0.001). This change was due to a change in amplitude, since the duration of the QRS complex did not alter significantly. The correlation between the changes in QRS-VD and body weight from the start to the end of the dialysis session was moderate and statistically significant (r = –0.55, p < 0.05). The correlation between the changes in QRS-VD and ECW varied from r = –0.67 to –0.97, being statistically significant in all patients (p < 0.001). The correlation between BV and QRS-VD was assessed at one minute intervals during the dialysis and varied from r = –0.22 to –0.98, being significant in 14 of the 15 patients (p < 0.001). Significant ST segments alterations (ST-VM6 elevation > 100 μV) did not occur during dialysis. Laboratory parameters reflecting volume and osmotic changes during hemodialysis correlated with QRS-VD change: B-Hcr (r = 0.56, p < 0.05), B-Hb (r = 0.63, p < 0.05), P-Na (r = 0.62, p < 0.05) and S-Urea (r = –0.62, p < 0.05). Conclusions: The increase in QRS complex amplitude during hemodialysis is correlated to reduced ECW. The mechanism involved is most probably augmentation of electrical resistance of the tissues around the heart caused by loss of interstitial fluid.