Axial fatigue experiments have been conducted employing notched samples of a 316L stainless steel under both constant and variable amplitude loading. A number of samples were subjected to high cycle fatigue (HCF) in the range of ∼144,000–271,000 cycles and subsequent tensile tests, in order to evaluate the change in tensile mechanical properties due to prior fatigue damage. The results obtained have shown that HCF in this range of cycles gives rise to a significant increase in the yield stress of the material, while other mechanical properties determined in tension remain almost constant. It has been determined that the increase in yield stress with the cyclic ratio ( D) is more significant as the maximum stress applied to the material increases. Such a behavior has been described by means of a simple parametric relationship, which could be employed for the practical determination of D in parts and components made of this material, by means of simple tensile testing. A plausible explanation of the increase in yield stress with D, based on the development of the dislocation substructure able to modify such a property, has been proposed. The fractographic analysis that has been conducted on the samples tested in tension after fatigue damage indicates that such a phenomenon also gives rise to the development of fatigue cracks, whose size increases with D. It has been shown that the number of cycles to fracture in two-stress step fatigue tests can be satisfactorily predicted by means of the Palmgren–Miner linear damage cumulative rule. A non-linear numerical procedure has been presented in order to re-compute the fatigue strength coefficient and fatigue exponent of the material, from data obtained under variable amplitude loading conditions, whose values give rise to a satisfactory description of the S–N experimental data. The use of more complex non-linear damage cumulative rules has not given rise to any significant improvement in the number of cycles recorded in the second stress step. It is believed that, due to the significant plastic deformation that occurs at the notch root during fatigue testing, cyclic creep (or ratcheting) damage in addition to fatigue damage could also be present.