The association between impairment in mitochondrial muscle fat oxidative capacity (OX(FA)) and occurrence of insulin resistance was examined in 14 healthy trained men (age, 24+/-4 yr) submitted to 4 weeks of training cessation. Training stop induced a significant decrease in mRNA levels of proteins involved in muscle fat metabolism, particularly PPARalpha (-58%, P<0.01) and PGC-1alpha (-30%, P<0.05), a 21% reduction in OX(FA) (P<0.01), and reduced fat oxidation during moderately intense exercise (P<0.05). In contrast, there was no significant alteration in insulin sensitivity. In conclusion, decline in OX(FA) is a rapid metabolic event following training cessation. It is involved in the regulation of whole body fat balance but not in the deterioration of insulin sensitivity.