We present a systematic microstructure-oriented plasticity investigation of an additively manufactured cobalt-based alloy aiming to relate microstructural changes to the fatigue resistance. A load-controlled cyclic test in the low-cycle fatigue regime was utilized to study mechanical response and microstructural changes in the alloy after reverse phase transformation heat treatment. Deformation-induced FCC → HCP phase transformation was followed using in situ synchrotron X-ray diffraction combined with ex situ electron backscatter diffraction and scanning electron microscopy. Scanning transmission electron microscopy provided experimental evidence of the presence of precipitates in the solution annealed sample. It was found, that a stepwise increase in plastic deformation is attributed to nucleation and growth of different martensitic crystallographic variants. These insights into plasticity and microstructural changes suggest that the reverse phase transformation heat treatment is an effective approach for improving the fatigue resistance of low stacking fault energy alloys, that are susceptible to deformation-induced martensitic transformation.