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During extracorporeal life support (ECLS), blood is exposed to a myriad of unphysiological factors that can affect outcome. One aspect of this is the sub-atmospheric pressure generated by the ECLS pump and imparted to blood elements along the pump inlet line. This pressure can be measured on the inlet line close to the pump head by adding a connector, or at the venous cannula connection site. We compared the two measurement sites located at both points; between the venous cannula-inlet tubing and inlet tubing-pump, with a range of cannulae and flows. We also investigated the effects on inlet pressure from pump afterload and increasing inlet tubing length.