This prospective single-center observational study compared impedance cardiography [electrical velocimetry (EV)] with transthoracic echocardiography (TTE, based on trans-aortic flow) and analyzed the influence of physiological shunts, such as patent ductus arteriosus (PDA) or patent foramen ovale (PFO), on measurement accuracy. Two hundred and ninety-one triplicate simultaneous paired left ventricular stroke volume (LVSV) measurements by EV (LVSV) and TTE (LVSV) in 99 spontaneously breathing neonates (mean weight 3270 g; range 1227-4600 g) were included. For the whole cohort, the mean absolute LVSV was 5.5 mL, mean LVSV was 4.9 mL, resulting in an absolute Bland-Altman bias of -0.7 mL (limits of agreement LOA -3.0 to 1.7 mL), relative bias -12.8 %; mean percentage error MPE 44.9 %; true precision TP 33.4 % (n = 99 aggregated data points). In neonates without shunts (n = 32): mean LVSV 5.0 mL, mean LVSV 4.6 mL, Bland-Altman bias -0.4 mL (LOA -2.8 to 2.0 mL), relative bias -8.2 %; MPE 50.7 %; TP 40.9 %. In neonates with shunts (PDA and/or PFO; n = 67): mean LVSV 5.8 mL, mean LVSV 5.0 mL, bias -0.8 mL (LOA -3.1 to 1.5 mL), relative bias -14.8 %, MPE 41.9 %, TP 29.3 %. Accuracy was affected by PDA and/or PFO, with a significant increase in the relative difference in LVSV versus LVSV: Subjects without shunts -2.9 % (n = 91), PFO alone -9.6 % (n = 125), PDA alone -14.0 % (n = 12), and PDA and PFO -18.5 % (n = 63). Physiological shunts (PDA and/or PFO) in neonates affect measurement accuracy and cause overestimation of LVSV compared with LVSV.