Current understanding of in vivo human brown adipose tissue (BAT) physiology is limited by a reliance on positron emission tomography (PET)/computed tomography (CT) scanning, which has measured exogenous glucose and fatty acid uptake but not quantified endogenous substrate utilization by BAT. Six lean, healthy men underwent 18fluorodeoxyglucose-PET/CT scanning to localize BAT so microdialysis catheters could be inserted in supraclavicular BAT under CT guidance and in abdominal subcutaneous white adipose tissue (WAT). Arterial and dialysate samples were collected during warm (∼25°C) and cold exposure (∼17°C), and blood flow was measured by 133xenon washout. During warm conditions, there was increased glucose uptake and lactate release and decreased glycerol release by BAT compared with WAT. Cold exposure increased blood flow, glycerol release, and glucose and glutamate uptake only by BAT. This novel use of microdialysis reveals that human BAT is metabolically active during warm conditions. BAT activation substantially increases local lipolysis but also utilization of other substrates such as glutamate. Display omitted •Human BAT shows substantial glucose uptake and lactate release during warm conditions•Glycerol recycling occurs in human BAT, and cold exposure activates BAT lipolysis•BAT uses substrates other than just glucose or fatty acids during cold activation•Microdialysis is a novel technique to measure in vivo human BAT physiology Weir et al., using microdialysis, have shown that human brown adipose tissue (BAT) is metabolically active in warm conditions, with higher glucose uptake and lactate release than white AT. Cold activation increased glucose and glutamate uptake and glycerol release by BAT, quantifying substrate utilization and hydrolysis of BAT triglycerides during thermogenesis.