The results show that human adaptive thermogenesis in response to cold exposure is accompanied by and related to mitochondrial uncoupling in skeletal muscle. Fifty percent of the variation in the individual increases in energy expenditure upon cold exposure can be explained by mitochondrial uncoupling
Since mitochondrial protein expression and citrate synthase activity did not increase after mild cold exposure, an augmentation of mitochondrial density can be ruled out 
. Also an increase in physical activity can be excluded, since radar counts were lower–and not higher–after cold exposure. Shivering can be excluded too; subjects reported they did not perceive any shivering at all. Furthermore, in a previous study in the same study setting, this has been validated by electromyogram measurements 
The process of skeletal muscle mitochondria uncoupling is thus likely to contribute to the increase in energy expenditure. Previously, it has been shown that mitochondrial uncoupling in humans could be stimulated. Infusion of T3, which is a known factor involved in cellular respiration, resulted in higher mitochondrial uncoupling 
. However, this study for the first time shows that environmental factors affect both adaptive thermogenesis and mitochondrial uncoupling.
The increase in energy expenditure, 0.32 MJ/day (−0.20 to 1.66 MJ/day), is comparable to data from literature 
. This increase is considerably smaller than the increase in energy expenditure observed in small mammals after cold exposure (two- to fourfold) 
. However, a small difference in energy expenditure for a prolonged period might contribute largely to weight gain. A diminished adaptive thermogenesis has indeed been identified as a risk factor for obesity 
. Therefore, our results indicate that mitochondrial uncoupling in human skeletal muscle may be a target for therapies to prevent obesity.
In the past, drugs have been developed to treat obesity, for example 2,4-Dinitrophenol, without insight in the mechanisms behind. This drug, which has a major effect on the uncoupling of oxidative phosphorylation, turned out to be lethal due to hyperthermia and has been banned in 1938, but is still used in illegal circuits due to its marked effects on body weight 
. The present study shows that skeletal muscle has the intrinsic capacity of regulated mitochondrial uncoupling. Research should aim to target this intrinsic uncoupling process in the mitochondrial membrane without disrupting the complete membrane as is the case with the use of 2,4-Dinitrophenol to increase thermogenesis and thus weight loss.
To gain more insight in the processes underlying cold-induced mitochondrial uncoupling, further research is needed to unravel the molecular nature of these processes in human skeletal muscle. Since UCP-1 and also the more recently discovered uncoupling proteins UCP-2, UCP-4, and UCP-5 are not present in human muscle 
, other candidate actors have to be found. UCP-3 is present in human muscle, but it is currently believed to play a role in fatty acid metabolism rather than in direct mitochondrial uncoupling 
. This is underlined by our finding that UCP-3 protein expression was not upregulated during cold exposure. Therefore, so far unknown proteins may be found that are able to provoke mitochondrial uncoupling. Proteomics and genomics approaches would be methods of choice, since with these methods no prior targets have to be determined.
Acclimatization to cold is known to have large effects on adaptive thermogenesis. Subjects regularly exposed to mild cold have a higher adaptive thermogenesis capacity 
. This could be of benefit in situations of overfeeding, since cold-induced adaptive thermogenesis is related to overfeeding induced adaptive thermogenesis, as we have shown recently 
. Therefore, the results indicate that regular cold exposure might be beneficial in body weight regulation via an increase in the skeletal muscle uncoupling capacity.
In conclusion, human skeletal muscle has the intrinsic capacity for mitochondrial uncoupling. Cold-induced increases in energy expenditure are accompanied by an increase in skeletal muscle mitochondrial uncoupling.