This study aimed to determine the effects of a single high-intensity interval training (HIIT) session on salivary cortisol (SC) levels, physiological responses, and performance in trained boys and men. Twenty-three boys (11.5 ± 0.8 years) and 25 men (29.7 ± 4.6 years) performed HIIT (4 consecutive Wingate Anaerobic Tests). SC in boys and men increased after HIIT from 5.55 ± 3.3 nmol/l to 15.13 ± 9.7 nmol/l (+173%) and from 7.07 ± 4.7 nmol/l to 19.19 ± 12.7 nmol/l (+171%), respectively (p < .01). Pretest SC as well as posttest changes were comparable in both groups (both p < .01). Peak blood lactate concentration was significantly lower in boys (12.6 ± 3.5 mmol/l) than in men (16.3 ± 3.1 mmol/l; p < .01). Throughout the HIIT, mean heart rates in boys were higher (p < .001) but relative peak oxygen uptake (ml·min−1·kg−1; p < .05) and performance were lower (p < .001) in boys than in men. HIIT in young athletes is associated with a higher activation of the hormonal stress axis than other types of exercise regimes as described in the literature. This study is the first to show a pronounced SC increase to HIIT in trained boys accompanied by elevated levels of blood lactate concentrations and heart rate suggesting a high cardio-respiratory, metabolic, and hormonal response to HIIT in 11-year-old boys.