Increased lipid delivery and disturbances in skeletal muscle lipid metabolism play a central role in the development of insulin resistance. In a previous cross-sectional study, we observed a reduced suppression of plasma fatty acids in response to an oral glucose tolerance test and increased dietary fat oxidation in middle-aged, but not older, patients with Type 2 diabetes (T2D). Elevated concentrations of several circulating lipid and branched chain amino acid (BCAA) derived metabolites, such as acylcarnitines (AC), have been consistently associated with T2D and ageing. However, their role in the development of skeletal muscle insulin resistance and T2D remains unclear. The aim of this study was to use a novel quantitative methodology to (a) investigate the association between T2D and a range of AC, BCAA and their respective keto acids (KA) in both plasma and human skeletal muscle from the volunteers in our previous study, and (b) examine to what extent ageing alters their profiles in T2D. Seven middle-aged (MA; 46.0 ± 1.1 years, BMI 29.6.1 ± 1.1) and 7 older-aged patients (OA; 63.4 ± 0.6, BMI 29.6 ± 1.3) with T2D, matched for disease duration, lean mass and metformin use, and 7 middle-aged normoglycemic controls (CON; 46.1 ± 2.9 years, BMI 31.1 ± 1.5) were recruited. Fasting vastus lateralis muscle biopsies and plasma samples were analysed for a range of individual AC, BCAA (leucine, isoleucine and valine) and their KA by liquid chromatography coupled to high resolution tandem mass spectrometry (LC-MS/MS) in a targeted approach. AC were grouped as short (C3-C5), medium (C6- C10) and long-chain (C12-C20). The muscle and plasma concentrations were quantified using an isotopically labelled internal standard method. Data are mean ± SEM and were compared by one-way ANOVA and Fisher’s LSD post hoc test where appropriate. Plasma short-chain AC (45 %, P < 0.01) and BCAA (17 %, P < 0.05) were higher in MA, but not OA vs. CON. Medium-chain AC were elevated (46 %, P < 0.05) in OA only. Inversely, the long-chain AC were lower in both MA (23 %, P < 0.05) and OA (22 %, P < 0.05) vs. CON. Skeletal muscle AC did not differ between groups. Muscle BCAA were elevated in MA (49%, P < 0.01) and OA (23 %, P < 0.05) vs. CON, with higher values in MA vs. OA (P < 0.05). There was no difference in plasma or muscle KA between groups. These findings reveal a lack of association between plasma and intramuscular concentrations of AC, suggesting that these species may not accurately reflect skeletal muscle lipid metabolism in both middle- and older-aged T2D patients. However, higher levels of plasma and muscle BCAA in MA than OA may better reflect perturbations in energy metabolism as indicated by higher dietary fat oxidation in the former group.