For vehicle electrification, one of the biggest issues for lithium ion batteries is cycle life. Within this context, the mechanisms at the source of capacity degradation during cycling are not yet to be fully understood. In this work, we use state-of-the-art FIB-SEM serial sectioning and imaging techniques to determine the effect of cycling on lithium-ion battery cathodes. The three-dimensional (3D) microstructural study was performed on both pristine and cycled LiNixMnyCo1−x−yO2 (NMC) and Li(Li0.2Ni0.13Mn0.54Co0.13)O2 (HE-NMC) cathodes. The spatial distribution of active material, carbon-doped binder and pore spaces were successfully reconstructed by appropriate image processing. Comparisons of NMC and HE-NMC cathodes after different number of cycles showed only minor increases in the number of smaller active particles, possibly negligible, considering the intrinsic microstructure variation within the cathodes. However, the connectivity between carbon-doped binder additives and active particles in NMC and HE-NMC cathodes, assessed using a “neighbor counting” method, showed an appreciable decrease after cycling which indicates a detachment of carbon-doped binder from active particles. This significant cycling-induced detachment effect between the two phases (e.g., ∼22% for HE-NMC) could indicate a loss in electrical connectivity, which may partially explain the capacity fade in the cells.