Pyruvate dehydrogenase (PDH) regulates oxidative carbohydrate disposal in skeletal muscle and is downregulated by reversible phosphorylation catalyzed by PDH kinase (PDK). Previous work has demonstrated increased PDK activity and PDK4 expression in human skeletal muscle following a high-fat low-carbohydrate (HF) diet, which leads to decreased PDH in the active form (PDHa activity) and carbohydrate oxidation. The purpose of this study was to examine the time course of changes in PDK and PDHa activities with refeeding of carbohydrates after an HF diet in human skeletal muscle. Healthy male volunteers ( n = 8) consumed a standardized 3-day Pre-diet with the same energy content as their habitual diet, followed by a eucaloric 6-day HF diet (Pre-diet: 50:30:20%; HF diet: 5:75:20%; carbohydrate/fat/protein). Muscle biopsies were taken before and after the HF diet and at 45 min and 3 h after carbohydrate refeeding with a single high-glycemic index carbohydrate meal (88:5:7% carbohydrate/fat/protein) representing approximately one third of the individual subject's habitual energy intake. PDK activity increased from 0.08 ± 0.01 Pre- to 0.25 ± 0.02 min ( P < 0.001) Post-HF diet, and decreased with carbohydrate refeeding to 0.17 ± 0.05 ( P = 0.014) and 0.11 ± 0.01 min ( P = 0.006) at 45 min and 3 h, respectively. PDHa decreased from 0.89 ± 0.20 to 0.32 ± 0.05 ( P = 0.007) mmol·min−1·kg wet wt−1 following the HF diet, and was increased transiently with refeeding at 45 min, but returned to lower values by 3 h ( P = 0.025 compared with Pre). The potential mechanism(s) for this attenuation of PDHa activity remains unclear. These data demonstrate that in human skeletal muscle, the adaptive increase in PDK activity following an HF diet is rapidly reversed to Pre-diet activity levels within 45 min to 3 h, and this is accompanied by a short-term increase in PDHa activity.