The role of food matrix and gender on soy isoflavone metabolism and biomarkers of activity were examined in twenty free-living adults (34.7 ± 11.5 years old) with hypercholesterolemia (221.9 ± 18.7mg dL(-1)). In a randomized crossover design study, participants consumed soy bread (3 wk) or a soy beverage (3 wk) containing 20 g soy protein with 99 and 93 mg isoflavones aglycone equivalents per day, respectively. During soy bread intervention, women had significantly greater microbial metabolite excretion (P = 0.05) of isoflavonoids than men. In men, isoflavone metabolite excretion was not discernibly different between the two matrices. Significant reductions (P≤ 0.05) in triglycerides (24.8%), LDL cholesterol (6.0%), apolipoprotein A-I (12.3%), and lipid oxidative stress capacity (25.5%), were observed after soy food intervention. Our findings suggest that the food matrix significantly impacts soy isoflavone metabolism, particularly microbial metabolites in women.