Abstract High-loaded tempered steels, being used in mobility, power generation and plant engineering, are frequently exposed to very high numbers of loading cycles (up to 1 billion cycles). Optimization and fatigue testing for such conditions is challenging and difficult, since damage is concentrated to very small areas of local cyclic plasticity. The concentration can be attributed to material inhomogeneities, such as segregations, large grains and non-metallic inclusions. The present paper demonstrates how the fatigue strength can be correlated with the material microstructure for two tempered steels, 50CrMo4 and 16MnCr7 7, respectively, in different heat-treatment conditions. This was shown by means of (i)in-situ damage observation during ultrasonic and resonance fatigue testing using light and scanning electron microscopy (SEM) in combination with high-resolution thermography, and (ii) quantitative analysis of internal VHCF crack initiation and the formation of fine granular areas (FGA) within the fracture surface using focused ion beam milling (FIB).