The fatigue damage accumulation behavior of GH4169 superalloy was studied through two-level loading fatigue tests based on an electromagnetic resonance testing machine. A total of 178 experimental data points and 13 S-N curves were obtained. By comparing and analyzing experimental data, the influence of stress and fatigue damage in the first-level loading on the residual fatigue life of the material was studied. The effect of first-level fatigue damage on the residual fatigue life of different second-level stresses was explained. A new fatigue damage accumulation rule was proposed and validated using several nonlinear fatigue cumulative damage models. The results indicate that this rule can serve as a criterion for evaluating the accuracy of fatigue cumulative damage models.