Recent studies have highlighted the importance of the biological clock as a modulator of energy balance and metabolism[1,2]. Intermittent fasting has become an increasingly popular intervention for metabolic health and combining intermittent fasting with exercise may lead to benefits for weight management[3]. However, little is known about the diurnal variation of fasted exercise and energy balance. Therefore, the aim of this study was to investigate the circadian influences on gastric emptying rate, blood glucose, and substrate utilisation during fasted and non-fasted brisk walking. Twelve healthy males (Mean ± SD; age 25 ± 3 years; height 178 ± 6 cm; body mass 83 ± 12 kg; VO2peak 39 ± 4 ml/kg/min) completed four 45 min treadmill walks in a randomised order. Walks were completed in the morning (AM) and evening (PM) and either fasted (FAST) or after consumption of a standardised breakfast meal (FED). Gastric emptying (GE) rate of a standardised semi-solid lunch meal was measured for 2 h using the 13C-breath method. Blood glucose concentration was measured at baseline, pre- and post-breakfast period, pre- and post-exercise, plus pre-lunch meal and at 30 min intervals following ingestion for 2 h. Substrate utilisation was measured every 30 min and continuously throughout exercise. Appetite was assessed at 15 min intervals throughout and 24 h energy intake post trials was assessed by diet diaries. No difference in GE half-emptying time was observed but lag phase was slower in PM-FAST compared to AM-FAST and PM-FED (75 ± 18 vs. 63 ± 14 min, P=0.006 and vs. 67 ± 16 min, P=0.040). Blood glucose concentration pre-exercise was greater in AM-FED compared to PM-FAST (5.80 ± 1.30 vs. 4.34 ± 0.31 mmol/L P=0.014), and greater in PM-FED compared to AM-FAST and PM-FAST (6.38 ± 1.15 vs. 4.69 ± 0.58, P=0.005 and 4.34 ± 0.31 mmol/L, P=0.001). No differences between trials were seen post-exercise (P>0.05), however, glucose concentration was greater pre-lunch in AM-FED compared to AM-FAST (5.28 ± 0.63 vs. 4.71 ± 0.40 mmol/L; P=0.042). Fat oxidation was greater pre-exercise in PM-FAST compared to PM-FED (0.11 ± 0.04 vs. 0.06 ± 0.02 g/min; P=0.003), greater throughout exercise for both FAST trials compared to FED (all P<0.05) and greater at pre-lunch in AM-FAST and PM-FAST than PM-FED (P=0.018; P=0.021). No differences between trials were observed for appetite post-lunch (P>0.05) or 24 h post-energy intake (P=0.476). These results suggest that appetite does not follow a diurnal variation following low intensity fasted exercise and that regardless of the time of day fasted exercise favours fat metabolism and may help induce a negative energy balance without a subsequent compensatory response in energy intake. Further studies on the implications of fasted exercise for energy balance and metabolic health are required.