Purpose of review The purpose of this review is to highlight recent methodological advances that have helped us to understand the role of intramuscular triglycerides in human health as well as prevention and treatment of disease. In addition to both invasive (muscle biopsy) and noninvasive methods, techniques to assess other lipids within muscle will be reviewed. Recent findings In addition to intramuscular triglycerides, other lipids contained within skeletal muscle such as diacylglycerol and ceramides may have an important role in human metabolic disease. Both ex-vivo and in-vivo methods to obtain serial measurements of these muscle lipids as a result of experimental perturbation have recently provided unique insight into their respective roles in human metabolism. Summary Triglyceride accumulation within skeletal muscle has received considerable interest due to its potential role in insulin resistance and fatty acid metabolism of obesity, aging and type 2 diabetes. Observations that triglycerides themselves may be used as a fuel source for exercising muscle, and thus may not be detrimental in all circumstances, has in part prompted the development and implementation of analytical methods to quantify the fatty acid composition of muscle triglycerides as well as other lipid species within muscle, e.g. diacylglycerol and ceramides. In addition, noninvasive computed tomography, magnetic resonance spectroscopy and magnetic resonance imaging methods have been applied to examine the serial effects of intervention on muscle triglycerides.