Increased adiposity is associated with insulin resistance, type 2 diabetes and metabolic dysfunction. Altered adipokine secretion, changes in local and systemic glucocorticoid metabolism and increased inflammation have all been postulated as contributory mechanisms. We have previously described changes in subcutaneous adipose tissue (SAT) gene expression that are associated with obesity and glucose intolerance in cross-sectional studies. However, the effects of longitudinal changes of gene expression to modulate metabolic phenotype have not been determined. 65 obese or overweight individuals (women=42) underwent oral glucose tolerance testing (OGTT), body composition analysis using DXA, 24-urinary steroid metabolome analysis and SAT biopsy. Participants were reinvestigated after a median of 5 years (IQR 3.55). Area under the curve glucose across the OGTT was used to stratify the cohort into those whose glucose tolerance improved over the duration of the study (improvers, n=30) and those in whom it deteriorated (deteriorators, n=35). SAT gene expression profiles (n=30) were determined using the Fluidigm platform. At baseline, there were no differences in fat mass or SAT gene expression profile. However, in the deteriorators, total body weight (91.5±17.1 vs 97.5±18.8 kg, P<0.0001), total fat mass (36325±8134 vs 39588±10190 g, P=0.0005) and trunk fat mass (18766±4786 vs 20753±5713 g, P<0.0001) increased, whilst these did not change significantly in the improvers. SAT gene expression profiles were markedly different between the two groups. In the deteriorators, the expression of genes associated with adipocyte lipid metabolism, including FAS (12.1±4.0 vs 10.4±1.9ΔCt, P=0.03), LPL (11.5±3.1 vs 9.6±1.5ΔCt, P=0.006), CD36 (9.4±2.8 vs 7.4±1.4ΔCt, P=0.002) and DGAT2 (11.6±3.5 vs 10.0±1.2, P=0.04) all increased during the study, whereas in the improvers, they remained unchanged. A similar pattern of expression was observed for those genes involved in insulin signaling, adipokine production and adipocyte differentiation. We have defined longitudinal changes in SAT gene expression that are associated with worsening glucose tolerance. This suggests a crucial role for SAT in the progressive development of glucose intolerance and highlights specific molecular markers that may drive this process.