Muscle pyruvate dehydrogenase kinase (PDK) plays an important role in the development of insulin resistance in metabolic states (eg starvation) that are characterised by elevated plasma free fatty acid (FFA) levels (Wu et al. 1999). However, the mechanism linking insulin resistance and altered PDK expression in skeletal muscle is not clear. The aim of this study was to investigate the effect of fasting and refeeding on insulin sensitivity and gene expression of skeletal muscle PDK isoforms, peroxisome proliferator-activated receptors (PPAR) α and δ (key regulators of fatty acid oxidation) and forkhead transcription factor FOXO1 (transcriptional activator of PDK4). Ten healthy male volunteers (age 26 ± 1 yr, BMI 25.5 ± 1.2 kg.m², mean ± SEM) participated in this study, which was approved by the local Ethics Committee. Subjects fasted for 48h (water, electrolytes and non-sugared beverages without caffeine were allowed only) and then consumed a high CHO diet (75% CHO, 10% fat and 15% protein) for 24h. Before and after fasting, and after refeeding, subjects underwent insulin tolerance tests (ITT) to quantify whole body insulin sensitivity. Muscle biopsies (vastus lateralis) were obtained in the fed state, after 24 and 48h of fasting, and after refeeding for the determination of PDK1-4, PPARα, PPARδ and FOXO1 mRNA by quantitative real-time PCR using Taqman probes and normalised to α-actin. Whole body insulin sensitivity (calculated from the rate constant for blood glucose disappearance during ITT) decreased by 42 ± 5% during fasting (P<0.01, 1-way ANOVA, relevant assumptions were verified) and recovered by 21 ± 7% (of initial value) upon refeeding. Fasting decreased blood glucose (P<0.01) and insulin (P<0.05) concentrations and increased plasma FFA levels (P<0.01) and muscle PDK4 mRNA content by 2.4-fold after 24h and 4.3-fold after 48h (P<0.05). Refeeding completely reversed those responses. There was no effect of fasting/refeeding on gene expression of PDK1-3 and FOXO1. In contrast, PPARα and PPARδ mRNA content decreased by ~2-fold after 48h of fasting (P<0.05) and returned to basal levels upon refeeding. In conclusion, starvation-induced insulin resistance was accompanied by a marked increase in PDK4 mRNA expression in human skeletal muscle, a response which did not involve changes in mRNA abundance of FOXO1 (as its expression remained unchanged during fasting/refeeding) or PPARα and PPARδ, suggesting that, in contrast to findings from animal studies, posttranslational modulations in key regulators of substrate oxidation mediate the shift in substrate utilisation from CHO to fat in fasted humans.