It is still unclear whether a high-intensity interval exercise (HIIE) protocol is effective for promoting anti-inflammatory and anti-atherogenic responses. The objective of this study was to analyze the acute effects of HIIE on inflammatory and metabolic responses in obese and overweight girls and boys. Initially, the body composition of 19 sedentary volunteers was evaluated. The participants completed an interval experimental run on a treadmill (2:1 min at 95% maximum velocity, totaling ~3 km) and blood samples were collected at rest and immediately after the exercise protocol. Serum was analyzed for interleukins 6 and 10 (IL-6 and IL-10), tumor necrosis factor-α (TNF-α), cortisol, glucose, triacylglycerol (TG), non-esterified fatty acid (NEFA), plasminogen activator inhibitor-1 (PAI-1), and the lipoprotein profile. Under baseline conditions, the groups of boys and girls exhibited differences in the anthropometry measures for fat-free mass ( p = 0.011), gynoid fat ( p = 0.002), visceral fat ( p = 0.004), and glucose ( p = 0.031). After the exercise session, cholesterol, glucose, NEFA, PAI-1, IL-10, and TNF-α did not demonstrate effects for time, group, or interaction under the different conditions. Cortisol (F = 9.018; p = 0.008; partial η2 = 0.347), TG (F = 25.189; p < 0.0001; partial η2 = 0.597), and IL-6 levels (F = 6.543; p = 0.020; partial η2 = 0.278) showed an effect for time. In conclusion, our results demonstrated that HIIE promoted an immune-endocrine response, increasing cortisol and IL-6 levels immediately after the exercise protocol in overweight and obese girls.