Abstract This paper describes the fatigue behavior of composite steel and concrete beams subjected to negative bending moment. Fatigue tests with repeated load limited to initial cracking and stabilized cracking, were performed on two steel–concrete composite beam substructures respectively. Test results indicated that when the repeated load was equivalent to the initial cracking load, the fatigue test had only limited influence on beam stiffness or crack patterns. However, when the repeated load was equivalent to the stabilized cracking load, a number of residual cracks occurred in the initial static test and the beam became less stiff as the load cycles increase. Failure of the bond between studs and surrounding concrete was confirmed in intermediate static tests; flexural stiffness of studs became smaller as the increase of load cycles. In addition, final static tests were performed on fatigue test specimens and another specimen without fatigue test. The comparison indicated that when the repeated load was larger than the initial cracking load, fatigue test could decrease beam stiffness and its ultimate load carrying capacity.