Mixing flow causes fluctuations in fluid temperature near a pipe wall and may result in fatigue crack initiation. Movement of the hot spot, at which the pipe inner surface was heated by hot flow from the branch pipe, causes thermal stress fluctuations. In this study, the effect of the loading sequence on thermal fatigue in a mixing tee was investigated. In addition, the prediction method of the fatigue life for the variable thermal strain in the mixing tee was discussed. The time histories of the strain around the hot spot were estimated by finite element analysis for which the temperature condition was determined by wall temperature measured in a mockup test. The accumulated fatigue damage around the hot spot 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. Crack growth tests showed that a single overload decreased crack opening strain and increased the effective strain range. The increment of the effective strain range accelerated the crack growth rate after the overload. The accumulated fatigue damage for the strain in the mixing tee was calculated using Miner's rule and the strain ranges, which added the maximum increment of the effective strain range. The accumulated fatigue damage was larger than 1.0 under most conditions. The proposed procedure is suitable to predict the conservative fatigue life in a mixing tee.