In long-distance running, 10–30% of the total training distribution of a macrocycle consists of high-intensity training, which effectively promotes performance. While training quality is compromised by hypoxia, high-intensity interval training (HIIT) performed under hypoxia elicits greater benefits than does normoxic training for lower-altitude athletes. However, its effects on moderate-altitude distance runners remain underexplored. This study aimed to compare the effects of HIIT performed at low (~1,220 m) and moderate (~2,850 m) altitudes on physiological, functional performance and perceptual responses, in moderate-altitude resident long-distance runners. To achieve this goal, forty-two runners (men/women, n=23/19) were randomly allocated into the HIIT group at ~2,850 m (HIIT2850m, n=14), the HIIT group at ~1,220 m (HIIT1220m, n=14), and the control group at ~2,850 m (CG2850m, n=14). While all participants maintained regular training at ~2,850 m, the HIIT groups completed two additional weekly sessions (4x4- minute at 100% vVO2 max, 3-minute recovery at 70% vVO2 max), lasting for 8 weeks. Before and after the intervention, measurements were performed during a group-based run on a 400 m track at ~2,850 m. The maximal aerobic capacity (VO2max) and the minimum running velocity associated with VO2max (vVO2max) were assessed via Cooper’s 12-minute test and 5-minute maximal aerobic speed running test, respectively. During the 5000-m race, the pace speed and perceived exertion were recorded every km. The results revealed significant changes in the main effect of time (p < 0.001) on the vVO2 max, VO2 max, and 5kRT in both intervention groups, with greater gains in the HIIT1220 m group (+1.2%, +1%, –1.8%) than in the HIIT2850 m group (+0.7%, +0.8%, –1.4%). In addition, a strong correlation was observed in the HIIT2850 m group between changes in 5kRT and vVO2 max (r = -0.6, p = 0.01) and VO2 max (r = 0.7, p = 0.002). The participants used a reversed parabolic-shaped strategy in both the group and time conditions, and a significant main effect of time (except at the 2nd km, p = 0.693) and group (at the 1st km, p = 0.034 and 5th km, p = 0.044) was observed. The results suggest that HIIT at both altitudes improved performance and RPE, with quality training at low altitudes producing greater benefits, offering strategic insights for athletes and coaches.