A novel in situ scanning electron microscope (SEM), high frequency fatigue testing methodology is developed using a combination of laser milling, focused ion beam fabrication and nanoindentation. This methodology is used to investigate crack initiation, propagation, fracture, fatigue life, and the mechanical response of microcantilever samples of a Ni-based superalloy (René-N5) under different cyclic strain amplitudes. The crack initiation and propagation in the microcantilever is monitored by observing changes in the beam's dynamic stiffness and continuous SEM imaging. The dynamic stiffness response of the micro-beams exhibits a transition from softening to hardening at a critical strain amplitude of 7×10−3. Theoretical analysis indicates that this transition corresponds to the stress required to shear γ′ precipitates. SEM imaging reveals the evolution of significant extrusions, intrusions, and slip traces during cyclic loading above this critical strain amplitude. Below this strain amplitude, very little surface roughening is observed. In addition, the measured dynamic stiffness is observed to exhibit two regimes of decrease after crack initiation. These two regimes correspond to short and large crack propagation. Finally, an overall increase in fatigue life is observed when comparing to bulk scale experiments on nickel-base superalloys. It is proposed that this is an inherent size effect in the small-volume, single crystal specimens tested.