It remains to be determined experimentally whether increasing fitness is related to positive selection, while stationary fitness is related to neutral evolution. Long-term laboratory evolution in Escherichia coli was performed under conditions of thermal stress under defined laboratory conditions. The complete cell growth data showed common continuous fitness recovery to every 2uC or 4 degrees C stepwise temperature upshift, finally resulting in an evolved E. coli strain with an improved upper temperature limit as high as 45.9 degrees C after 523 days of serial transfer, equivalent to 7,560 generations, in minimal medium. Two-phase fitness dynamics, a rapid growth recovery phase followed by a gradual increasing growth phase, was clearly observed at diverse temperatures throughout the entire evolutionary process. Whole-genome sequence analysis revealed the transition from positive to neutral in mutation fixation, accompanied with a considerable escalation of spontaneous substitution rate in the late fitness recovery phase. It suggested that continually increasing fitness not always resulted in the reduction of genetic diversity due to the sequential takeovers by fit mutants, but caused the accumulation of a considerable number of mutations that facilitated the neutral evolution.