Brown adipose tissue (BAT) plays a key role in adaptive thermogenesis in mammals, and it has recently been considered as an attractive therapeutic target for tackling human obesity by increasing energy expenditure. Thermal imaging using infrared thermography (IRT) has emerged as a potential safe, rapid and inexpensive technique for detecting BAT in humans. However, little attention has been given to the reliability of this method in obese subjects. To this end, we evaluated the capacity of IRT to detect activated supraclavicular (SCV) BAT in 14 lean and 16 mildly obese young adults after acute cold exposure. Using IRT we measured the temperature of the skin overlying the SCV and sternal areas at baseline and after acute cold stimulation. Additionally, energy expenditure was measured by indirect calorimetry and body composition was estimated using bioelectrical impedance analysis. Energy expenditure and SCV skin temperature significantly increased in lean subjects upon cold exposure, while no significant changes were detected in the obese group. Furthermore, cold-induced variations in SCV skin temperature of obese subjects showed a negative correlation with body mass index. This study suggests that in lean individuals BAT is a rapidly activated thermogenic tissue possibly involved in the regulation of energy balance, and can be indirectly assessed using IRT. In obese subjects, BAT seems less prone to be activated by cold exposure, with the degree of adiposity representing a limiting factor for the indirect detection of BAT activation by measuring the skin temperature overlying BAT.