Chronic complications observed in patients with long-chain 3-hydroxyacylCoA dehydrogenase (LCHAD) or trifunctional protein (TFP) deficiency may be mediated by the accumulation of 3-hydroxy fatty acids or 3-hydroxyacylcarnitines. To understand variation in metabolite accumulation, their concentrations were measured by tandem mass spectrometry before and after a mixed meal and moderate intensity exercise. Subjects who were homozygous or heterozygous for the common mutation (c.1528G>C) in the TFP alpha subunit (LCHAD deficiency) had significantly higher 3-hydroxyacylcarnitines than subjects with TFP deficiency. Feeding a mixed meal significantly suppressed and exercise significantly increased plasma 3-hydroxyacylcarnitines concentrations.