The introduction of ¹³C magnetic resonance spectroscopy (MRS) has enabled noninvasive measurement of muscle glycogen synthesis in humans. Conclusions based on measurements by the MRS technique assume that glucose metabolism in gastrocnemius muscle is representative for all skeletal muscles and thus can be extrapolated to whole-body muscle glucose metabolism. An alternative method to assess whole-body muscle glycogen synthesis is the use of [3-³H]glucose. In the present study, we compared this method to the MRS technique, which is a well-validated technique for measuring muscle glycogen synthesis. Muscle glycogen synthesis was measured in the gastrocnemius muscle of six lean healthy subjects by MRS and by the isotope method during a hyperinsulinemic-euglycemic clamp. Mean muscle glycogen synthesis as measured by the isotope method was 115 ± 26 μmol · kg⁻¹ muscle · min⁻¹ vs. 178 ± 72 μmol · kg⁻¹ muscle · min⁻¹ (P = 0.03) measured by MRS. Glycogen synthesis rates measured by MRS exceeded 100% of glucose uptake in three of the six subjects. We conclude that glycogen synthesis rates measured in gastrocnemius muscle cannot be extrapolated to whole-body muscle glycogen synthesis.