Aims: to measure whether subjects with high (HBAT) and low activation of brown adipose tissue (LBAT) showed acute differences in energy expenditure during and after 30-minutes of high intense interval training. Methods: Forty professional soccer athletes were invited to participate in the study, of which 37 started the protocol and 35 completed it. This was an acute, double-blind, case-control, quasi-experimental study design. Activation of BAT was estimated to be using infrared thermography. All participants were divided into four groups and compared by condition: (a) HBAT-CAF; (b) HBAT-PLA; (c) LBAT-CAF; and (d) LBAT-PLA. They performed 30 min of high-intensity interval training (HIIT) which included four sets of 4-minute of running in a speed corresponding at 90–95% of HRmax, each followed by a 3-minute active recovery interval at 60–70% of HRmax and 30 min of passive recovery. During exercise and recovery, breath-to-breath spirometry was performed and energy expenditure (EE), carbohydrates (g/day), fats (g/day) and proteins (g/day) were measured. Results: HBAT showed higher EE at all measurement times (p≤0.01) in the CAF condition and at all measurement times in the PLA condition (p≤0.001). For fat catabolism, the CAF condition showed a significant difference between conditions, with CAF showing higher catabolism at 60 min [36.5 g/day (0.8; 137.2); p=0.047] compared to PLA. For PTN catabolism, the HBAT-CAF condition, minutes 40, 50, and 60 showed a significant difference compared to the other measurement times (p≤0.023 for all comparisons). Conclusion: After high-intensity interval exercise with CAF supplementation, both HBAT and LBAT subjects experience a significant increase in EE during exercise and recovery compared to baseline. Basal BAT activity did not affect the rate of CHO and FAT oxidation, although a higher rate of PTN utilization was observed in the HBAT-CAF group.