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Based on the engineering background of a new type of segmental-assembled steel temporary beam buttress, the fatigue strength evaluation method of the steel box girders with open longitudinal ribs was taken as the research objective. The fatigue stress calculation analysis and the full-scale fatigue loading test for the steel box girder local component were carried out. The accuracy of the finite-element model was verified by comparing it with the test results, and the rationality of the fatigue strength evaluation methods for welded joints was deeply explored. The results indicate that the maximum nominal stress occurs at the weld toe between the transverse diaphragm and the top plate at the edge of the loading area, which is the fatigue-vulnerable location for the steel box girder local components. The initial static-load stresses at each measuring point were in good agreement with the finite-element calculation results. However, the static-load stress at the measuring point in the fatigue-vulnerable position shows a certain decrease with the increase in the number of cyclic loads, while the stress at other measuring points remains basically unchanged. According to the finite-element model, the fatigue strengths obtained by the nominal stress method and the hot-spot stress method are 72.1 MPa and 93.8 MPa, respectively. It is reasonable to use the nominal stress S-N curve with a fatigue life of 2 million cycles at 70 MPa and the hot-spot stress S-N curve with a fatigue life of 2 million cycles at 90 MPa (FAT90) to evaluate the fatigue of the welded joints in steel box girders with open longitudinal ribs. According to the equivalent structural stress method, the fatigue strength corresponding to 2 million cycles is 94.1 MPa, which is slightly lower than the result corresponding to the main S-N curve but within the range of the standard deviation curve. The research results of this article can provide important guidance for the anti-fatigue design of welded joints in steel box girders with open longitudinal ribs. Full article