The evolution of the substructure and the distribution of interstitial elements in lath martensite during tempering in soft martensitic stainless steel X4CrNiMo16-5-1 was studied with line profile analysis of diffractograms from energy dispersive synchrotron X-ray diffraction, local chemical analysis with atom probe tomography and orientation mapping with electron backscatter and transmission Kikuchi diffraction. Martensite formation occurred below 135 °C without auto-tempering and led to a dislocation density in martensite of 3.8 • 10 15 m −2 , as determined from X-ray line profile analysis. On tempering, carbon and nitrogen segregated to low-angle and high-angle grain boundaries. Recovery commenced above 550 °C and led to a reduction in dislocation density to a steady value of 4 • 10 14 m −2 from 600 to 750 °C. Further tempering led to a second increase in dislocation density at room temperature, owing to the transformation of reverted austenite, formed above 650 °C, into martensite on cooling. It was observed that the recovery of martensite competes with the formation of reverted austenite. The interpretation of the coherently diffracting domain size obtained from X-ray line profile analysis was critically discussed in the context of the internal structure in martensite.