Adult-onset growth hormone deficiency (GHD) is associated with insulin resistance and decreased exercise capacity. Intramyocellular lipids (IMCL) depend on training status, diet and insulin sensitivity. Using magnetic resonance spectroscopy (MRS), we studied IMCL content following physical activity (IMCL-depleted) and high fat diet (IMCL-repleted) in 15 patients with GHD before and after 4 months of GH replacement therapy (GHRT) and in 11 healthy control subjects. Measurements of insulin resistance and exercise capacity were performed and skeletal muscle biopsies were carried out to assess expression of mRNA of key enzymes involved in skeletal muscle lipid metabolism using real-time PCR and ultrastructure using electron microscopy. Compared with control subjects, patients with GHD showed significantly higher delta-IMCL (difference between IMCL-depleted and IMCL-repleted). GHRT resulted in an increase in skeletal muscle mRNA expression of IGF-I, hormone sensitive lipase and a tendency for an increase in fatty acid binding protein-3. Electron microscopy examination did not reveal significant differences after GHRT. In conclusion, variation of IMCL may be increased in patients with GHD compared to healthy control subjects. Qualitative changes within the skeletal muscle (i.e. an increase in free fatty acids availability from systemic and/or local sources) may contribute to the increase in insulin resistance and possibly to the improvement of exercise capacity after GHRT. The upregulation of IGF-I mRNA suggests a paracrine/autocrine role of IGF-I on skeletal muscle.