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Detecting and assessing changes in the hydrologic cycle and its response to a changing environment is essential for maintaining regional ecological security and restoring degraded ecosystems. There is no clear scientific evidence on the effects of human activities and climate variability on runoff and its components in typical arid areas. Therefore, in this study, a heuristic segmentation algorithm, a variable infiltration capacity model (VIC), and remote sensing data to quantify the effects of human activities and climate variability on runoff in the catchment of Lake Ebinur, Xinjiang, China. The results found: (1) The heuristic segmentation algorithm divided the study period into reference period (1964–1985) and two impact periods: I (1986–2000) and II (2001–2017). (2) Cropland and forest land showed an increasing trend, with grassland and barren land accounting for most of the increase. At the same time, the leaf area index (LAI) increased by 0.002 per year during the growing season. (3) Compared with the reference period, runoff depth decreased by 108.80 mm in impact period I due to human activities, but increased by 110.5 mm due to climate variability, resulting in an overall increase in runoff depth of 1.72 mm. Runoff depth increased by 11.10 mm in the impact period II compared to the reference period, with climate variability resulting in an increase of 154.40 mm, but human activities resulted in a decrease of 143.30 mm. Our results shed light on decision-making related to water stress in changing circumstances in arid regions. [ABSTRACT FROM AUTHOR]