Fasting in human subjects shifts skeletal muscle metabolism toward lipid utilization and accumulation, including intramyocellular lipid (IMCL) deposition. Growth hormone (GH) secretion amplifies during fasting and promotes lipolysis and lipid oxidation, but it is unknown to which degree lipid deposition and metabolism in skeletal muscle during fasting depends on GH action. To test this, we studied nine obese but otherwise healthy men thrice: (a) in the postabsorptive state (“CTRL”), (b) during 72-hr fasting (“FAST”), and (c) during 72-hr fasting and treatment with a GH antagonist (GHA) (“FAST + GHA”). IMCL was assessed by magnetic resonance spectroscopy (MRS) and blood samples were drawn for plasma metabolomics assessment while muscle biopsies were obtained for measurements of regulators of substrate metabolism. Prolonged fasting was associated with elevated GH levels and a pronounced GHA-independent increase in circulating medium- and long-chain fatty acids, glycerol, and ketone bodies indicating increased supply of lipid intermediates to skeletal muscle. Additionally, fasting was associated with a release of short-, medium-, and long-chain acylcarnitines to the circulation from an increased β-oxidation. This was consistent with a ≈55%–60% decrease in pyruvate dehydrogenase (PDHa) activity. Opposite, IMCL content increased ≈75% with prolonged fasting without an effect of GHA. We suggest that prolonged fasting increases lipid uptake in skeletal muscle and saturates lipid oxidation, both favoring IMCL deposition. This occurs without a detectable effect of GHA on skeletal muscle lipid metabolism.