Recently developed confocal microscopes allow image acquisition at rapid frame-rates (e.g. 120 frames per second for images of size 512 by 512 pixels) and open new avenues for cardiac imaging at the microscopic scale. The reconstruction and analysis of dynamic 3D data of embryonic hearts require further image processing. The main challenges are the handling of the considerable amount of data generated for each experiment and the need for a reliable and repeatable analysis procedure. Here we present the workflow for the reconstruction of 4D volumetric data from nongated 2D image sequences acquired in living zebrafish embryos and the subsequent data analysis to extract atrial and ventricular volume changes over time. The former is performed using a wavelet-based synchronization procedure while the latter is made possible via semi-automatic segmentation of the atrial and ventricular heart regions.