The purpose of this in vitro study was to evaluate the impact of the vertical level of the stopcock connecting the infusion line to the central venous catheter on start-up fluid delivery in microinfusions. Start-up fluid delivery was measured under standardized conditions with the syringe outlet and liquid flow sensors positioned at heart level (0 cm) and exposed to a simulated CVP of 10 mmHg at a set flow rate of 1 ml/h. Flow and intraluminal pressures were measured with the infusion line connected to the stopcock primarily placed at vertical levels of 0 cm, + 30 cm and − 30 cm or primarily placed at 0 cm and secondarily, after connecting the infusion line, displaced to + 30 cm and − 30 cm. Start-up fluid delivery 10 s after opening the stopcock placed at zero level and after opening the stopcock primarily connected at zero level and secondary displaced to vertical levels of + 30 cm and – 30 cm were similar (− 10.52 [− 13.85 to − 7.19] µL; − 8.84 [− 12.34 to − 5.33] µL and − 11.19 [− 13.71 to − 8.67] µL (p = 0.469)). Fluid delivered at 360 s related to 65% (zero level), 71% (+ 30 cm) and 67% (− 30 cm) of calculated infusion volume (p = 0.395). Start-up fluid delivery with the stopcock primarily placed at + 30 cm and − 30 cm resulted in large anterograde and retrograde fluid volumes of 34.39 [33.43 to 35.34] µL and − 24.90 [− 27.79 to − 22.01] µL at 10 s, respectively (p < 0.0001). Fluid delivered with the stopcock primarily placed at + 30 cm and − 30 cm resulted in 140% and 35% of calculated volume at 360 s, respectively (p < 0.0001). Syringe infusion pumps should ideally be connected to the stopcock positioned at heart level in order to minimize the amounts of anterograde and retrograde fluid volumes after opening of the stopcock.