A multi-layered thin film structure (namely, electrodeposited Cu/sputtered Cr/Kapton substrate/sputtered Cr/electrodeposited Cu), utilized as a flexible component for computers, has been exposed to fatigue. Although a standardized testing method for fatigue ductility is available for a solid monolayer of electrodeposited foil, there is no method available for examining such a multi-layered thin film structure. In this study, four different methods were employed to characterize the low-cycle fatigue damage: (1) DC resistance measurement, (2) residual stress development by x-ray diffraction, (3) dislocation density calculation by using obtained x-ray diffraction line profiles, and (4) microscopic observations. Low-cycle fatigue was conducted at eight levels of applied total strain, i.e., δe T =13.95%, 7.69%, 5.83%, 4.69%, 3.37%, 2.37%, 1.59%, and 1.19%. The number of fatigue cycles, when the crack was first observed on the outer Cu layer, was identical to that observed with the onset of increased resistance. This cycle number is thus designated as the number of cycles-to-fatigue crack initiation,N c . AtN c , the residual stresses also show a noticeable relaxation, and the dislocation density shows a remarkable increase. IfN c is plotted against the applied total strain amplitudes, a Manson-Coffin's relationship is obtained with an exponent of 0.39. It is recommended that monitoring the continuous changes in DC resistance could provide a reliable nondestructive evaluation of low-cycle fatigue life of a multi-layered thin film structure.