To understand the day-to-day pathophysiology of impaired muscle glycogen storage in type 2 diabetes, glycogen concentrations were measured before and after the consumption of sequential mixed meals (breakfast: 190.5 g carbohydrate, 41.0 g fat, 28.8 g protein, 1,253 kcal; lunch: 203.3 g carbohydrate, 48.1 g fat, 44.0 g protein, 1,497.5 kcal) by use of natural abundance ¹³C magnetic resonance spectroscopy. Subjects with diet-controlled type 2 diabetes (n = 9) and age- and body mass index-matched nondiabetic controls (n = 9) were studied. Mean fasting gastrocnemius glycogen concentration was significantly lower in the diabetic group (57.1 ± 3.6 vs. 68.9 ± 4.1 mmol/l; P < 0.05). After the first meal, mean glycogen concentration in the control group rose significantly from basal (97.1 ± 7.0 mmol/l at 240 min; P = 0.005). After the second meal, the high level of muscle glycogen concentration in the control group was maintained, with a further rise to 108.0 ± 11.6 mmol/l by 480 min. In the diabetic group, the postprandial rise was markedly lower than that of the control group (65.9 ± 5.2 mmol/l at 240 min, P < 0.005, and 70.8 ± 6.7 mmol/l at 480 min, P = 0.01) despite considerably greater serum insulin levels (752.0 ± 109.0 vs. 372.3 ± 78.2 pmol/l at 300 min, P = 0.013). This was associated with a significantly greater postprandial hyperglycemia (10.8 ± 1.3 vs. 5.3 ± 0.2 mmol/l at 240 min, P < 0.005). Basal muscle glycogen concentration correlated inversely with fasting blood glucose (r = 0.55, P < 0.02) and fasting serum insulin (r = 0.57, P < 0.02). The increment in muscle glycogen correlated with initial increment in serum insulin only in the control group (r = 0.87, P < 0.002). This study quantitates for the first time the subnormal basal muscle glycogen concentration and the inadequate glycogen storage after meals in type 2 diabetes.