Mixing flow causes fluid temperature fluctuations near the pipe walls and may result in fatigue crack initiation. The authors have previously reported the loading sequence effect on thermal fatigue in a mixing tee. The fatigue damage around the hot spot, which was heated by the hot jet flow from the branch pipe, obtained by Miner’s rule was less than 1.0. Since the strain around the hot spot had waveforms with periodic overload, the loading sequence with periodic overload caused reduction of the fatigue life around the hot spot. In this study, the effect of a single overload on the fatigue crack growth rate was investigated in order to clarify the reduction of the fatigue life at the mixing tee due to strain with periodic overload. In addition, the prediction method of the fatigue life for the variable thermal strain at the mixing tee was discussed. It was shown the crack growth rate increased after an overload for both cases of tensile and compressive overloads. The effective strain amplitude increased after the application of a single overload. The fatigue life curve was modified by considering the increment of the effective strain range. The fatigue damage recalculated using the modified fatigue life curve was larger than 1.0 except in a few cases. The fatigue life could be assessed conservatively for variable strain at the mixing tee using the developed fatigue curve and Miner’s rule.