**Background:** High LDL cholesterol is a major risk factor for atherosclerotic cardiovascular disease (ASCVD), while HDL cholesterol shows an inverse relationship with ASCVD risk. However, standard lipid measurements cannot differentiate between lipoprotein subfractions that vary in particle size, density, and composition, many of which have contrasting relationships with ASCVD risk. Small dense LDL particles are associated with incident ASCVD independent of total LDL cholesterol, while only the largest HDL subclasses were inversely associated with myocardial infarction risk in large cohort studies. Lifestyle modification remains the cornerstone of ASCVD prevention, but the effects of high-fat diets and exercise timing on detailed lipoprotein subfraction profiles are not well understood.
**Methods:** This was a secondary analysis of a randomised trial with three parallel groups conducted at Australian Catholic University. Twenty-four men (mean age 36±4 years, BMI 31.2±2.3 kg/m²) consumed a high-fat diet (HFD) consisting of 65% total energy intake (TEI) from fat (52% saturated, 10% polyunsaturated, 38% monounsaturated), 15% from carbohydrate, and 20% from protein for 11 days. After 5 days, participants were randomised to no exercise (CONTROL, n=8), morning exercise at 06:30h (EXam, n=8), or evening exercise at 18:30h (EXpm, n=8) on days 6-10. Exercise consisted of high-intensity interval training (10×1-min work bouts at 95-120% peak power output) on days 6, 8, and 10, and moderate-intensity continuous cycling (40-60 min at 60-65% peak power output) on days 7 and 9. Venous blood samples were collected fasting (morning) and postprandial (evening) at baseline, after 5 days of HFD, and at study completion. Lipoprotein subclassification was performed using Bruker IVDr Lipoprotein Subclass Analysis (B.I.LISA™) based on 600 MHz NMR spectroscopy, providing concentrations of cholesterol, free cholesterol, phospholipids, triglycerides, Apo-A1, Apo-A2, and Apo-B across 100 subfraction variables (VLDL 1-5, IDL, LDL 1-6, HDL 1-4). Statistical analyses included principal component analysis (PCA), multilevel partial least squares discriminant analysis (PLS-DA), repeated measures ANOVA simultaneous component analysis (RM-ASCA+), and univariate linear mixed models with Benjamini-Hochberg adjustment for multiple comparisons (q<0.05 considered significant).
**Key Results:** Five days of HFD induced significant changes in 31 of 100 lipid variables in fasting samples and 41 variables in postprandial samples. Fasting total serum VLDL cholesterol was reduced by approximately 25% (q=0.039), with significant reductions in larger VLDL particles (VLDL-1-3). Total fasting serum LDL cholesterol increased due to increased cholesterol in larger LDL particles (LDL-2 and LDL-3), with a shift in Apo-B distribution from small (LDL-6) to larger LDL subfractions (LDL-2 and LDL-3) without significant change in total LDL Apo-B. HDL-related variables showed reduced triglyceride enrichment in HDL-3 and HDL-4. Exercise training induced significant decreases in 20 lipid variables after EXam and 24 after EXpm (15 in common). Compared with CONTROL, EXpm reduced fasting cholesterol concentrations in LDL-1 (q=0.002), LDL-3 (q=0.044), and LDL-4 (q=0.013) by approximately 30%, while EXam only reduced LDL-1 cholesterol (q=0.020) by 19%. Both exercise groups reduced cholesterol concentrations in HDL-3 and HDL-4 (q<0.05 for both). There were no statistically significant differences between EXam and EXpm for any fasting or postprandial lipoprotein subfraction variables. Classification models separated pre- and post-HFD samples with 79% accuracy (fasting) and 100% accuracy (postprandial).
**Clinical Implications:** The HFD-induced shift toward larger, more buoyant LDL particles and reductions in large VLDL subfractions may be interpreted as mostly beneficial for cardiometabolic health, given that small dense LDL particles and large VLDL particles are more strongly associated with ASCVD risk and insulin resistance. The reduction in triglyceride enrichment in small HDL particles after HFD also suggests potential benefit, as triglyceride concentrations in small HDL are associated with myocardial infarction risk. Exercise training, regardless of time of day, further improved the lipoprotein profile by reducing concentrations in large LDL particles and small HDL particles. The study is limited by its short duration (11 days), small sample size (n=24), inclusion of only men, and inherent differences in post-exercise sampling timing between morning and evening exercise groups (12 vs 24 hours for fasting samples). The authors caution that these short-term experimental findings should not be taken as clinical recommendations, particularly given ongoing scientific debate about the relationship between dietary fat and ASCVD.