Metal films on flexible polymer substrates are commonly used in flexible electronic devices and may be exposed to repeated large deformations during manufacturing and application. To ensure a long life time of flexible electronics, these compound systems have to be robust and reliable while stretching and compressing without failing mechanically or electrically. The goal of this work was to examine the influence of microstructure and film thickness on the electro-mechanical deformation behaviour of copper-polymer film-substrate systems. Therefore, four different copper films on 50 μm polyimide substrates with variation in thickness (50 nm, 100 nm and 200 nm) and grain size due to annealing were tested. The microstructure of the Cu films was determined using electron backscatter diffraction, focused ion beam and transmission electron microscopy. The choice of method depended on the thickness of the investigated film. The average grain size was found to be approximately in the range of the film thickness. The 50 nm and the 100 nm thick films only possessed nano-sized grains, the as-deposited and annealed 200 nm thick Cu films exhibited large micron-sized grains. The deformation behaviour of the substrate bonded Cu films was determined by in-situ tensile tests, both mechanically with fragmentation tests under the atomic force microscope and electrically with 4 point probe resistance measurements. The onset of plastic deformation in the form of localized thinning at the surface was detected at low strains for all film thicknesses, whereas for films with coarser grains and larger film thicknesses necks formed earlier. Although the deformation density rose with increasing strain for all samples, the electrical conductivity of the Cu films did not deteriorate exceedingly for the 100 nm and 200 nm films. The 50 nm films, however, showed a great deterioration of the conductivity due to the formation of cracks. In conclusion, Cu films with a film thickness under 100 nm showed a rather brittle deformation behaviour caused by their nano-sized grains. Above 100 nm film thickness ductile deformation behaviour was observed. Instead of cracks only necks formed in the film during straining, which could be attributed to the presence of large grains in the range of microns.