Aims/hypothesis Individuals of South Asian origin are at increased risk of developing type 2 diabetes mellitus and associated comorbidities compared with Europids. Disturbances in energy metabolism may contribute to this increased risk. Skeletal muscle and possibly also brown adipose tissue (BAT) are involved in human energy metabolism and nitric oxide (NO) is suggested to play a pivotal role in regulating mitochondrial biogenesis in both tissues. We aimed to investigate the effects of 6 weeks of supplementation with l -arginine, a precursor of NO, on energy metabolism by BAT and skeletal muscle, as well as glucose metabolism in South Asian men compared with men of European descent. Methods We included ten Dutch South Asian men (age 46.5 ± 2.8 years, BMI 30.1 ± 1.1 kg/m 2 ) and ten Dutch men of European descent, that were similar with respect to age and BMI, with prediabetes (fasting plasma glucose level 5.6–6.9 mmol/l or plasma glucose levels 2 h after an OGTT 7.8–11.1 mmol/l). Participants took either l -arginine (9 g/day) or placebo orally for 6 weeks in a randomised double-blind crossover study. Participants were eligible to participate in the study when they were aged between 40 and 55 years, had a BMI between 25 and 35 kg/m 2 and did not have type 2 diabetes. Furthermore, ethnicity was defined as having four grandparents of South Asian or white European origin, respectively. Blinding of treatment was done by the pharmacy (Hankintatukku) and an independent researcher from Leiden University Medical Center randomly assigned treatments by providing a coded list. All people involved in the study as well as participants were blinded to group assignment. After each intervention, glucose tolerance was determined by OGTT and basal metabolic rate (BMR) was determined by indirect calorimetry; BAT activity was assessed by cold-induced 18 Ffluorodeoxyglucose ( 18 FFDG) positron emission tomography–computed tomography scanning. In addition, a fasting skeletal muscle biopsy was taken and analysed ex vivo for respiratory capacity using a multisubstrate protocol. The primary study endpoint was the effect of l -arginine on BAT volume and activity. Results l -Arginine did not affect BMR, 18 FFDG uptake by BAT or skeletal muscle respiration in either ethnicity. During OGTT, l -arginine lowered plasma glucose concentrations (AUC 0–2 h  − 9%, p  < 0.01), insulin excursion (AUC 0–2 h  − 26%, p  < 0.05) and peak insulin concentrations (−26%, p  < 0.05) in Europid but not South Asian men. This coincided with enhanced cold-induced glucose oxidation (+44%, p  < 0.05) in Europids only. Of note, in skeletal muscle biopsies several respiration states were consistently lower in South Asian men compared with Europid men. Conclusions/interpretation l -Arginine supplementation does not affect BMR, 18 FFDG uptake by BAT, or skeletal muscle mitochondrial respiration in Europid and South Asian overweight and prediabetic men. However, l -arginine improves glucose tolerance in Europids but not in South Asians. Furthermore, South Asian men have lower skeletal muscle oxidative capacity than men of European descent. Funding This study was funded by the EU FP7 project DIABAT, the Netherlands Organization for Scientific Research, the Dutch Diabetes Research Foundation and the Dutch Heart Foundation. Trial registration: ClinicalTrials.gov NCT02291458.