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Changes in loading position have a significant impact on the stress field of each vulnerable area of an orthotropic steel deck (OSD). The arc opening area of the diaphragm and the connecting area between the U-rib and the diaphragm under the moving load are prone to fatigue cracking. By comparing the stress responses under different methods, the hot spot stress (HSS) method is used as the main stress extraction method in fatigue performance evaluation. The control stress of fatigue cracking was analyzed by comparing the direction of the principal stress field with the crack direction in this experiment. According to the stress amplitude deviation under the biaxial stress state, a set of methods for evaluating the effects of in-plane biaxial fatigue was developed. An improved luffing fatigue assessment S-N curve was applied to analyze the fatigue life of the diaphragm's arc opening area. The results show that when the moving load is exactly above the connection of the deck and the web of the U-rib on one side, it is in the most unfavorable position in the transverse direction, and the diaphragm is mainly under the in-plane stress state. The longitudinal range of the stress influence line of the arc opening is approximately twice the diaphragm spacing. Two to three stress cycles are caused by one fatigue load. Fatigue crack control stress is the principal stress tangential to the arc opening's edge in this area. The normal direction of the principal stress in the model test is roughly consistent with the crack initiation direction. The variation in the stress amplitude deviation in this area is caused by changes in the action position of the moving load. When the moving load is at a certain distance from the involved diaphragm, it is reduced to zero, implying that the in-plane fatigue effect is the greatest in this area.