The arterio-venous balance (A-V balance/difference) technique has been used by a number of groups, including ours, to study skeletal muscle fatty acid metabolism. Several lines of evidence indicate that like glycogen, intramyocellular triglycerides (imcTG) are an energy source for local use. As such, the report that increased release of free fatty acids (FFA) via lipolysis from skeletal muscle, but not from adipose tissue, is responsible for the increased systemic lipolysis during IL-6 infusion in healthy humans is somewhat unexpected (26). It appears that given the complex anatomy of human limbs, as to be discussed below, it is virtually impossible to determine whether any fatty acids being released into the venous circulation of an arm or leg derive from the lipolysis of intermuscular fat residing between muscle groups, intramuscular fat residing within muscle groups (between epimysium and perimysium, or bundles), or from the intramyocellular triglyceride droplets (imcTG). In many cases, it may be even difficult to be confident there is no contribution of FFA from subcutaneous adipose tissue. This question is fundamentally important as one attempts to interpret the results of skeletal muscle fatty acid metabolism studies using the A-V balance technique. In this mini review, we examine the reported results of fatty acid kinetics obtained using the techniques to evaluate the degree of and how to minimize contamination when attempting to sample skeletal muscle-specific fatty acids.