Lipids are stored not only in adipocytes but also ‘ectopically’ in tissues such as muscle, liver, beta cells and others. From a metabolic perspective, intramyocellular lipids (IMCLs) have recently become a focus of interest. This review summarizes history, measurement techniques and interpretation of muscle lipid data. Problems in biopsies with the separation of those metabolically active lipid droplets in the cytoplasm of myocytes from further lipids in adipocytes are discussed as well as considerations important for analysis of correlations between IMCL content and insulin sensitivity under various circumstances. The relatively new approach to non-invasive assessment of the IMCL content by magnetic resonance spectroscopy (MRS) is described in detail and exemplary spectra from different skeletal muscle types in humans are presented. The MRS technique allows human examinations of large cohorts for a detailed assessment of the interactions among metabolic parameters such as age, measures of adiposity, hormonal and ethnic factors and insulin resistance. IMCLs are generally positively correlated with measures of obesity and negatively with insulin sensitivity. Paradoxically, physical fitness (maximal aerobic capacity) increases both IMCL content and insulin sensitivity and therefore has to be taken into account as a confounding factor. Intervention studies with MRS further allowed to elucidate the regulation of IMCL. Molecular mechanisms and potential genetic factors on IMCL regulation are discussed as well as possible mechanisms of current treatment strategies for improving insulin sensitivity.