Carbon-13 magnetic resonance spectroscopy (13C MRS) offers a noninvasive method to assess glycogen levels in skeletal muscle and to identify excess glycogen accumulation in patients with glycogen storage disease (GSD). Despite the clinical potential of the method, it is currently not widely used for diagnosis or for follow-up of treatment. While it is possible to perform acceptable 13C MRS at lower fields, the low natural abundance of 13C and the inherently low signal-to-noise ratio of 13C MRS makes it desirable to utilize the advantage of increased signal strength offered by ultra-high fields for more accurate measurements. Concomitant with this advantage, however, ultra-high fields present unique technical challenges that need to be addressed when studying glycogen. In particular, the question of measurement reproducibility needs to be answered so as to give investigators insight into meaningful inter-subject glycogen differences. We measured muscle glycogen levels in vivo in the calf muscle in three patients with McArdle disease (MD), one patient with phosphofructokinase deficiency (PFKD) and four healthy controls by performing 13C MRS at 7T. Absolute quantification of the MRS signal was achieved by using a reference phantom with known concentration of metabolites. Muscle glycogen concentration was increased in GSD patients (31.5±2.9 g/kg w. w.) compared with controls (12.4±2.2 g/kg w. w.). In three GSD patients glycogen was also determined biochemically in muscle homogenates from needle biopsies and showed a similar 2.5-fold increase in muscle glycogen concentration in GSD patients compared with controls. Repeated inter-subject glycogen measurements yield a coefficient of variability of 5.18%, while repeated phantom measurements yield a lower 3.2% system variability. We conclude that noninvasive ultra-high field 13C MRS provides a valuable, highly reproducible tool for quantitative assessment of glycogen levels in health and disease.