In this study, four-dimensional (3D+t) ultrasound imaging techniques were used for the development and in vivo verification of 3D strain imaging. Two different iterative coarse-to-fine 3D strain estimation methods were developed. One method was based on measuring displacements using 3D kernels and a 3D cross-correlation function. The second method used 2D kernels and cross-correlation, and estimated 3D displacements in an iterative process. A 3D or 2D parabolic interpolation was used for sub-sample displacement estimates. The strain estimation methods were experimentally validated using a gelatin phantom with a hard cylindrical inclusion (four times stiffer). The phantom was compressed with a plate in steps of 0.5 mm up to 3.0 mm (3% strain). Rf-data were acquired with a 3D matrix array transducer (X4, Philips Sonos 7500) in ECG-triggered 3D full volume mode. Preliminary in vivo validation was performed by acquiring 3D full volume data (frame rate = 19 Hz) of the left ventricle of a trained athlete. Both methods were able to produce high quality elastograms of the inclusion model up to an applied compression of 3% strain (resulting in 0.5% - 5% axial strain in the phantom). No significant difference in elastographic signal-to-noise ratio (SNRe) was found between the two methods. The iterative 2D algorithm is favored for the shorter computation time. The signal- and contrast-to-noise ratios (SNRe, CNRe) of the axial elastograms increased to 28 and 53 dB, respectively (compared to previously described BiPlane axial strain images). Lateral and elevational elastograms were also in accordance with finite element solutions of the phantom model. However, the SNRe and CNRe were considerably lower (16 and 33 dB), which is presumably caused by the lower in-plane spatial resolution of the 3D full volume data. Initial in vivo results revealed mean strain profiles in three orthogonal directions comparable with our previous studies, although, the maximum radial strain was lower than expected (20%). Hence, 3D cardiac strain imaging is feasible even at a relatively low frame rate.