Due to their high cost efficiency and flexibility, semi-precast concrete slabs with lattice girders are widely used in constructions all over the world. Prefabricated concrete slabs, combined with in situ concrete topping, exhibit a quasi-monolithic structural behavior in which lattice girders serve as vertical shear reinforcement and ensure the transfer of longitudinal shear within the interface, acting in combination with concrete-to-concrete bonding mechanisms. To be applicable in industrial and bridge construction, semi-precast slabs need to have sufficient resistance against fatigue failure. To improve and expand the limits of application, theoretical and experimental investigations are conducted at the Institute of Structural Concrete (IMB), RWTH Aachen University. To investigate the fatigue behavior of lattice girders, small size tests with lattice girder diagonals were carried out. These test results have been used to derive an S–N curve (S: stress, N: number of load cycles) for lattice girders for a more refined fatigue design. Subsequently, the fatigue behavior of semi-precast slabs with lattice girders was investigated by fatigue tests on single-span slab segments. The fatigue design regulations of lattice girders according to technical approvals can generally be confirmed by this test program; however, they tend to be conservative. The use of the derived S–N curve leads to significantly improved agreement of fatigue behavior observed in tests and design expressions.