Mechanical forces can regulate various functions in living cells. The cytoskeleton is a crucial element for the transduction of forces in cell-internal signals and subsequent biological responses. Accordingly, many studies in cellular biomechanics have been focused on the role of the contractile acto-myosin system in such processes. A widely used method to observe the dynamic actin network in living cells is the transgenic expression of fluorescent proteins fused to actin. However, adverse effects of GFP-actin fusion proteins on cell spreading, migration and cell adhesion strength have been reported. These shortcomings were shown to be partly overcome by fusions of actin binding peptides to fluorescent proteins. Nevertheless, it is not understood whether direct labeling by actin fusion proteins or indirect labeling via these chimaeras alters biomechanical responses of cells and the cytoskeleton to forces. We investigated the dynamic reorganization of actin stress fibers in cells under cyclic mechanical loading by transiently expressing either egfp-Lifeact or eyfp-actin in rat embryonic fibroblasts and observing them by means of live cell microscopy. Our results demonstrate that mechanically-induced actin stress fiber reorganization exhibits very different kinetics in EYFP-actin cells and EGFP-Lifeact cells, the latter showing a remarkable agreement with the reorganization kinetics of non-transfected cells under the same experimental conditions.