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Background: The methods of estimating vascular access (VA) flow rates are usually based on the indicator dilution theory by measuring recirculation during dialysis sessions. Methods: This is an observational study comparing the VA flow rates measured by NIKKISO DBB-EXA™ and Transonic®. Sixty-five patients (38 M/27 F, mean age 72 ± 10 years) participated in the study. We measured the VA flow rates during dialysis twice with each method and repeated the procedure 7 days later. Results: In 130 double measurements for each method on the same day, mean flow with Transonic® was 1413±715 ml/min and with DBB-EXA™ 1297 ± 664 ml/min. In Bland-Altman analysis, the mean difference between the two methods was 159 ± 211 ml/min (limits of agreement: −274 and 572 ml/min). Eighty-one out of the 130 DBB-EXA™ measurements were within 25% of the Transonic® measurements (62% accuracy). Regarding reproducibility of each method on different days, mean difference in the Bland-Altman analysis was 29 ± 620 ml/min (limits of agreement: −1186 and 1244 ml/min) for the Transonic® measurements and 132 ± 625 ml/min (limits of agreement: −1092 and 1356 ml/min) for the DBB-EXA™ measurements. The measurements on two different days were within 25% of each other for 52 of the 65 patients (80%) with the Transonic® method, and for 35 of the 65 patients (54%) with the DBB-EXA™ method. Conclusions: In conclusion, the DBB-EXA™ method underestimates VA flow rates compared to the Transonic® technique, resulting in a limited accuracy of 62%. There was poor reproducibility for both methods in different day measurements with better performance of the Transonic® technique. The DBB-EXA™ method could be used as a simple tool for a rough estimate of VA flow rates but cannot replace the Transonic® reference method.