BackgroundComparing four-dimensional flow against two-dimensional flow measurements in patients with complex flow pattern is still lacking. This study aimed to compare four-dimensional against the two-dimensional flow measurement in patients with bicuspid aortic valve and to test potentials of four-dimensional operator-dependent sources of error.ResultsThe two- and four-dimensional flow data sets of sixteen patients with bicuspid aortic valve and eighteen healthy subjects were studied. Flow analyses were performed by two observers blindly. Patients with bicuspid aortic valve mean differences between the two- and four-dimensional measurements in both observers were − 8 and − 4 ml, respectively. Four-dimensional measurements resulted in systematically higher flow values than the two-dimensional flow in bicuspid aortic valve patients. The upper and lower limits of agreement between the two- and four-dimensional measurements by both observers were + 12/− 28 ml and + 14/− 21 ml, respectively. In the healthy volunteers, mean differences between the two- and four-dimensional measurements in both observers were ± 0 and + 1 ml, respectively. The upper and lower limits of agreement between the two- and four-dimensional measurements by both observers were + 21/− 18 ml and + 12/− 13 ml, respectively. Inter-observer variability in four-dimensional flow measurement was 4% mean net forward flow in bicuspid aortic valve patients and 8% in healthy volunteers.ConclusionInter-observer variability in four-dimensional flow assessment is 8% or less which is acceptable for clinical cardiac MRI routine. There is close agreement of two- and four-dimensional flow tools in normal and complex flow pattern. In complex flow pattern, however, four-dimensional flow measurement picks up 4–9% higher flow values. It seems, therefore, that four-dimensional flow is closer to real flow values than two-dimensional flow, which is however to be proven by further studies.