AbstractAimTo investigate how delayed post‐exercise carbohydrate intake affects muscle glycogen, metabolic‐ and mitochondrial‐related molecular responses, and subsequent high‐intensity interval exercise (HIIE) capacity.MethodsIn a double‐blind cross‐over design, nine recreationally active men performed HIIE (10 × 2‐min cycling, ~94% W˙_peak) in the fed state, on two occasions. During 0–3 h post‐HIIE, participants drank either carbohydrates (“Immediate Carbohydrate” [IC], providing 2.4 g/kg) or water (“Delayed Carbohydrate” [DC]); total carbohydrate intake over 24 h post‐HIIE was matched (~7 g/kg/d). Skeletal muscle (sampled pre‐HIIE, post‐HIIE, +3 h, +8 h, +24 h) was analyzed for whole‐muscle glycogen and mRNA content, plus signaling proteins in cytoplasmic‐ and nuclear‐enriched fractions. After 24 h, participants repeated the HIIE protocol until failure, to test subsequent HIIE capacity; blood lactate, heart rate, and ratings of perceived effort (RPE) were measured throughout.ResultsMuscle glycogen concentrations, and relative changes, were similar between conditions throughout (p > 0.05). Muscle glycogen was reduced from baseline (mean ± SD mmol/kg dm; IC: 409 ± 166; DC: 352 ± 76) at post‐HIIE (IC: 253 ± 96; DC: 214 ± 82), +3 h (IC: 276 ± 62; DC: 269 ± 116) and + 8 h (IC: 321 ± 56; DC: 269 ± 116), returning to near‐baseline by +24 h. Several genes (PGC‐1ɑ, p53) and proteins (p‐ACC^Ser79, p‐P38 MAPK^{Thr180/Tyr182}) elicited typical exercise‐induced changes irrespective of condition. Delaying carbohydrate intake reduced next‐day HIIE capacity (5 ± 3 intervals) and increased RPE (~2 ratings), despite similar physiological responses between conditions.ConclusionMolecular responses to HIIE (performed in the fed state) were not enhanced by delayed post‐exercise carbohydrate intake. Our findings support immediate post‐exercise refueling if the goal is to maximize next‐day HIIE capacity and recovery time is ≤24 h.