This study aimed to determine whether glucose-fructose (GF) ingestion, relative to glucose-only, would alter performance, metabolism, gastrointestinal (GI) symptoms, and psychological affect during prolonged running. On two occasions, 20 runners (14 men) completed a 120-min submaximal run followed by a 4-mile time trial (TT). Participants consumed glucose-only (G) or GF (1.2:1 ratio) beverages, which supplied ˜ 1.3 g/min of carbohydrate. Substrate use, blood lactate, psychological affect [Feeling Scale (FS)], and GI distress were measured. Differences between conditions were assessed using magnitude-based inferential statistics. Participants completed the TT 1.9% (−1.9; −4.2, 0.4) faster with GF, representing a likely benefit. FS ratings were possibly higher and GI symptoms were possibly-to-likely lower with GF during the submaximal period and TT. Effect sizes for GI distress and FS ratings were relatively small (Cohen's d = ˜0.2 to 0.4). GF resulted in possibly higher fat oxidation during the submaximal period. No clear differences in lactate were observed. In conclusion, GF ingestion – compared with glucose-only – likely improves TT performance after 2 h of submaximal running, and GI distress and psychological affect are likely mechanisms. These results apply to runners consuming fluid at 500–600 mL/h and carbohydrate at 1.0–1.3 g/min during running at 60–70% VO2peak.