PurposeThis study investigated whether the FLASH effect could be triggered using compact single high-energy X-ray source (CHEXs) FLASH radiotherapy (FLASH-RT) or single gantry rotation short-interval fractional irradiation in mice.Experimental DesignThe absolute dose and pulsed beam of the CHEXs were measured using an EBTXD radiochromic film and fast current transformer. Healthy C57BL/6J female mice and a subcutaneous tumor model were irradiated under different conditions: sham (control), FLASH-RT (FLASH1 and FLASH3, delivering the total dose in 1 or 3 fractions, with a 30-s interval between fractions), and conventional dose rate radiotherapy (CONV-RT). Various total doses were administered to the corresponding normal tissues (whole body, 9 Gy; whole thorax, 30 Gy; whole abdomen, 12 Gy; and skin, 36 Gy) and tumors (B16-F10, 24 Gy; CT26, 16 Gy; and LLC, 18 Gy). Survival status, normal tissue damage, and tumor growth suppression were recorded in each group.ResultsThe average dose rate of the CHEXs exceeded 40 Gy/s. For whole-body irradiation, the white blood cell count in the FLASH-RT group was significantly higher than that in the CONV-RT group (P<0.05). For whole-thorax and skin irradiation, both FLASH1 and FLASH3 demonstrated protective effects. For whole-abdomen irradiation, FLASH1 exhibited a superior protective effect. No significant differences in tumor growth responses were observed between the FLASH1, FLASH3, and CONV-RT groups (P>0.05).ConclusionBoth CHEXs and single-gantry rotation short-interval fractional irradiation can trigger the FLASH effect. This suggests that CHEXs may be beneficial for three-dimensional conformal radiotherapy.