Abstract: Objective To study the pattern of DNA double-strand break (DSB) formation in S-phase cells after thermal damage and explore the mechanisms behind heat sensitivity of S-phase cells and delayed DSBs. Methods Flow cytometry was used to analyze the cell cycle arrest in H1299 cells exposed to thermal damage, and EdU incorporation assay was employed to evaluate the DNA replication capacity of the cells. The cells synchronized in S phase were obtained by serum starvation and DSBs were observed dynamically using neutral comet assay. Trypan blue dye exclusion technique was used to analyze the cell viability after thermal damage. Western blotting (WB) was used to detect the phosphorylation of ATM and DNA binding RAD18. Results The percentage of S-phase cells increased significantly after exposure of the cells to 45 ℃ for 1 h (P<0.01). The time-dependent variation pattern of EdU incorporation was similar to that of S-phase cell fraction. The comet tail began to appear only after incubation of the cells at 37 ℃ for some time and the Olive tail moment (OTM) increased with prolonged incubation. Cell death remained low until 7.5 h after heat exposure of the S-phase cells and then increased rapidly. The phosphorylation of ATM first increased but then decreased drastically. In cells with heat exposure, DNA binding RAD18 was attenuated obviously compared that in non-exposed cells. Conclusion Thermal damage causes cell cycle arrest in S phase, and delayed fatal DSBs occur in the arrested cells due to persistent replication and DNA damage repair suppression, which are the possible cause of heat sensitivity of S-phase cells.