This study aimed to describe the acute impact of extended interval training (EIT) on physiological and thermoregulatory levels, as well as to determine the influence of athletic performance and age effect on the aforementioned response in endurance runners. Thirty-one experienced recreational male endurance runners voluntarily participated in this study. Subjects performed EIT on an outdoor running track, which consisted of 12 runs of 400 m. The rate of perceived exertion, physiological response through the peak and recovery heart rate, blood lactate, and thermoregulatory response through tympanic temperature, were controlled. A repeated measures analysis revealed significant differences throughout EIT in examined variables. Cluster analysis grouped according to the average performance in 400 m runs led to distinguish between athletes with a higher and lower sports level. Cluster analysis was also performed according to age, obtaining an older group and a younger group. The one-way analysis of variance between groups revealed no significant differences (p≥0.05) in the response to EIT. The results provide a detailed description of physiological and thermoregulatory responses to EIT in experienced endurance runners. This allows a better understanding of the impact of a common training stimulus on the physiological level inducing greater accuracy in the training prescription. Moreover, despite the differences in athletic performance or age, the acute physiological and thermoregulatory responses in endurance runners were similar, as long as EIT was performed at similar relative intensity.