Purpose This study aimed to examine the effects of reduced CHO but high postexercise fat availability on cell signaling and expression of genes with putative roles in regulation of mitochondrial biogenesis, lipid metabolism, and muscle protein synthesis.

Methods Ten males completed a twice per day exercise model (3.5 h between sessions) comprising morning high-intensity interval training (8 × 5 min at 85% V˙O 2peak ) and afternoon steady-state (SS) running (60 min at 70% V˙O 2peak ). In a repeated-measures design, runners exercised under different isoenergetic dietary conditions consisting of high-CHO (HCHO: 10 g·kg −1 CHO, 2.5 g·kg −1 protein, and 0.8 g·kg −1 fat for the entire trial period) or reduced-CHO but high-fat availability in the postexercise recovery periods (HFAT: 2.5 g·kg −1 CHO, 2.5 g·kg −1 protein, and 3.5 g·kg −1 fat for the entire trial period).

Results Muscle glycogen was lower ( P < 0.05) at 3 h (251 vs 301 mmol·kg −1 dry weight) and 15 h (182 vs 312 mmol·kg −1 dry weight) post-SS exercise in HFAT compared with HCHO. Adenosine monophosphate-activated protein kinase α2 activity was not increased post-SS in either condition ( P = 0.41), although comparable increases (all P < 0.05) in PGC-1α, p53, citrate synthase, Tfam, peroxisome proliferator-activated receptor, and estrogen-related receptor α mRNA were observed in HCHO and HFAT. By contrast, PDK4 ( P = 0.003), CD36 ( P = 0.05), and carnitine palmitoyltransferase 1 ( P = 0.03) mRNA were greater in HFAT in the recovery period from SS exercise compared with HCHO. Ribosomal protein S6 kinase activity was higher ( P = 0.08) at 3 h post-SS exercise in HCHO versus HFAT (72.7 ± 51.9 vs 44.7 ± 27 fmol·min −1 ·mg −1 ).