This study aimed to examine performance and ratings of physical perceived exertion (P-RPE) and task effort awareness (TEA) during a steady state and 20 km cycling time trial when muscle glycogen concentration at the start of cycling was either “normal” or “high” and during which euglycaemia was maintained throughout by means of a glucose clamp technique. After performing a muscle glycogen depletion protocol, cyclists (n = 8) were given, on two separate occasions and in random order, a diet designed to maintain low muscle glycogen concentration which was consumed for 3 days, during with a high carbohydrate or placebo drink was ingested to either maintain glycogen at a low concentration or carbohydrate load the cyclists before the performance tests. Cyclists subsequently rode in the laboratory on a Computrainer™ cycle-ergometer at 60% Wpeak for an hour, followed immediately by a 20 km time trial. Heart rate, P-RPE, TEA, power output (W) and RER data were collected throughout the trials. Euglycaemia was maintained at 4.5–5.5 mmol/L throughout both the steady state and 20km time trial via a glucose clamp technique. There were no significant differences in HR, P-RPE, or TEA during the steady state ride, however CHO oxidised was significantly higher (p = 0.014) in the high vs low glycogen condition (3.63 ± 0.59 vs 2.24 ± 1.28 g/min). There were no differences in the 20 km time trial in performance times (33:40 ± 1:05 (mean ± SD) vs 33:22 ± 0:52 min:sec), power output (242 ± 20 vs 247 ± 16W) or heart rate (164 ± 7 vs 164 ± 8 beats/min) in the low vs high starting muscle glycogen condition, respectively. It is concluded that low muscle glycogen concentration per se is therefore not the cause of, or perception of, fatigue during prolonged exercise. Rather, as long as euglycaemia is maintained, working muscle can continue to produce adequate force despite glycogen depletion.