Background: Durability of plate fixation is important in delayed union. Although locking plates result in stronger constructs, it is not known if locking affects the fatigue life of a plate. Two locking screws on either side of the nonunion could decrease working length and increase strain in the plate. However, the reinforcing effect of the locking head on the plate may compensate, so that it is unclear whether locking reduces fatigue life. Questions/purposes: We determined whether locking screws, compression screws, and locking buttons reduce or increase the fatigue life of a plate. Methods: We tested fatigue life of four constructs using an eight-hole locking plate in a segmental defect model: (1) all locking screws (Locked; n = 5); (2) all compression screws (Unlocked; n = 5); (3) six compression screws with two locking buttons in the central holes (Button; n = 6); and (4) six compression screws with two open central holes (Open; n = 6). Results: The Button group had the longest fatigue life (1.3 million cycles). There was no difference between the Locked and Unlocked groups. All of the constructs failed by fracture of the plates through a screw hole adjacent to the defect. Conclusions: Locking screws did not improve fatigue life, however a locking button increased the fatigue life of a locking plate in a segmental bone defect model. Clinical Relevance: Locking buttons in holes adjacent to a defect may improve durability, which is important when delayed union is a possibility.