Prior moderate exercise reduces plasma triglyceride (TG)-rich lipoprotein concentrations, mainly in the large very low-density lipoprotein (VLDL₁) fraction, but the mechanism responsible is unclear. We investigated the effects of brisk walking on TG-rich lipoprotein kinetics using a novel method. Twelve overweight/obese middle-aged men underwent two kinetic studies, involving infusion of Intralipid to block VLDL₁ catabolism, in random order. On the afternoon prior to infusion, subjects either walked on a treadmill for 2 h at ∼50% maximal oxygen uptake or performed no exercise. Multiple blood samples were taken during and after infusion for separation of Intralipid (S(f) 400) and VLDL₁ (S(f) 60-400). VLDL₁-TG and -apoB production rates were calculated from their linear rises during infusion; fractional catabolic rates (FCR) were calculated by dividing linear rises by fasting concentrations. Intralipid-TG FCR was determined from the postinfusion exponential decay. Exercise reduced fasting VLDL₁-TG concentration by 30% (P = 0.007) and increased TG enrichment of VLDL₁ particles [30% decrease in cholesteryl ester (CE)/TG ratio (P = 0.007); 26% increase in TG/apoB ratio (P = 0.059)]. Exercise also increased VLDL₁-TG, VLDL₁-apoB, and Intralipid-TG FCRs by 82, 146, and 43%, respectively (all P < 0.05), but had no significant effect on VLDL₁-TG or -apoB production rates. The exercise-induced increase in VLDL₁-apoB FCR correlated strongly with the exercise-induced changes in VLDL₁ CE/TG (r = -0.659, r = 0.020) and TG/apoB (r = 0.785, P = 0.002) ratios. Thus, exercise-induced reductions in VLDL₁ concentrations are mediated by increased catabolism, rather than reduced production, which may be facilitated by compositional changes to VLDL₁ particles that increase their affinity for clearance from the circulation.