Muscle Glycogen Analysis

On collection, muscle biopsy samples were immediately dissected free of visible fat and connective tissue and frozen in liquid nitrogen, before being stored at −70°C and subsequently powdered and freeze-dried. 2–5 mg of dry muscle (DM) tissue was hydrolyzed by adding 500 µL of 1 mmol·L⁻¹ HCl and subsequently incubated for 2 h in an oven at 95°C. After cooling to room temperature, samples were neutralized with 2 M NaOH. This was followed by centrifugation (1,800 g at 4°C for 10 min) and the supernatant was analyzed in duplicate for glucose using an automated photometric-based clinical chemistry analyzer (described below). Where tissue size permitted (∼50%), muscle glycogen concentration was determined in duplicate. The intraassay coefficient of variation for muscle glycogen determination was <10%.