Purpose: To define the effect of glucose ingestion compared to sucrose ingestion on liver and muscle glycogen depletion during prolonged endurance-type exercise. Methods: Fourteen cyclists completed two 3-h bouts of cycling at 50% of peak power output while ingesting either glucose or sucrose at a rate of 1.7 g/min (102 g/h). Four cyclists performed an additional third test in which only water was consumed for reference. We employed 13C magnetic resonance spectroscopy to determine liver and muscle glycogen concentrations before and after exercise. Expired breath was sampled during exercise to estimate whole-body substrate use. Results: Following glucose and sucrose ingestion, liver glycogen levels did not show a significant decline following exercise (from 325±168 to 345±205 and 321±177 to 348±170 mmol/L, respectively; P>0.05) with no differences between treatments. Muscle glycogen concentrations declined (from 101±49 to 60±34 and 114±48 to 67±34 mmol/L, respectively; P<0.05), with no differences between treatments. Whole-body carbohydrate utilization was greater with sucrose (2.03±0.43 g/min) vs glucose ingestion (1.66±0.36 g/min; P<0.05). Both liver (from 454±33 to 283±82 mmol/L; P<0.05) and muscle (from 111±46 to 67±31 mmol/L; P<0.01) glycogen concentrations declined during exercise when only water was ingested. Conclusion: Both glucose and sucrose ingestion prevent liver glycogen depletion during prolonged endurance-type exercise. Sucrose ingestion does not preserve liver glycogen concentrations more than glucose ingestion. However, sucrose ingestion does increase whole-body carbohydrate utilization compared to glucose ingestion. This trial was registered at clinicaltrials.gov as NCT02110836.