In cardiac MRI, the evaluation of left ventricular ejection fraction (LVEF) is based on well-defined protocols and involves a stack of 2D cine images acquired during multiple breath-holds. This procedure is time-consuming and may result in suboptimal coverage and inter-slice misregistration. Here, a novel acquisition protocol for 3D cine imaging with whole-heart coverage in one single breath-hold with isotropic spatial resolution is described and tested in 10 subjects in comparison to the 2D gold-standard. All 2D datasets and 9/10 3D datasets were considered adequate for LVEF calculation. Bland-Altman analysis of LVEF shows good agreement and no bias between the two measurements.