Background <p>Women with prior gestational diabetes mellitus (GDM) have an elevated risk of developing cardiometabolic diseases, including type 2-diabetes and cardiovascular disease. While physical fitness is protective, circulating molecular biomarkers linking fitness to long-term metabolic health in this population remain poorly understood. This study aimed to identify serum proteins associated with aerobic capacity and muscle strength 10 years after GDM, and explore their biological functions related to cardiometabolic risk.</p> Methods <p>We assessed aerobic fitness (VO₂peak), peak fat oxidation, and maximal isometric muscle strength of five muscle groups in 38 women from the post-GDM PONCH-cohort. Serum proteins were analysed using mass spectrometry-based proteomics. Associations between proteins, fitness variables, and clinical markers were tested using Spearman correlations with FDR correction, and age- and medication-adjusted sensitivity analysis. Group differences across four fitness-level groups (defined by aerobic fitness and muscle strength) and glycaemic status groups were analysed using linear regression models and Kruskal-Wallis tests, with age- and medication-adjusted sensitivity analyses. Exercise responsiveness of selected proteins was assessed in an independent cohort of untrained men undergoing six weeks of supervised aerobic training (<i>n</i> = 28), with pre-post changes assessed using Wilcoxon signed-rank tests.</p> Results <p>Thirty-five proteins were associated with at least one fitness variable, of which 21 remained significant after age- and medication-adjusted sensitivity analysis. Nine proteins correlated with both VO₂peak and muscle strength. Identified proteins mapped to key cardiometabolic pathways, including metabolic regulation, immune response, complement activation, oxidative stress, and extracellular matrix remodelling. Five proteins (PON3, IGF1, CRISP3, COL6A3, C3) emerged as particularly interesting, showing the largest and most consistent effect sizes across fitness variables and associations with central adiposity, blood lipids, blood pressure, and insulin resistance. Stratified analysis across the four fitness-level groups identified IGF1, PON3, and PRG4 as markers of higher overall-fitness. In the independent training cohort, nine of the fitness-associated proteins changed following aerobic training without significant weight-loss.</p> Conclusions <p>This study identified circulating proteins linked to physical fitness and cardiometabolic health in women after GDM. These findings suggest fitness-associated serum proteins may serve as biomarkers of early metabolic dysfunction and potential targets for exercise-based prevention of T2D and cardiovascular disease.</p> Graphical abstract <p></p>