Objectives: The present study evaluates the effect of food intake on 90 biomarkers for cardiovascular disease (CVD) and inflammation with the Proseek Multiplex CVD II kit. Methods: Twenty-two healthy subjects (11 male and 11 females aged 25.9±4.2 years) were investigated. A total of 90 biomarkers were measured before a standardized meal, 30 and 120 minutes thereafter with the Proseek Multiplex CVD II kit. Results: The levels for 27 biomarkers changed significantly after food intake. Two biomarkers increased 120 minutes after food intake, five biomarkers decreased 30 minutes after food intake, seven biomarkers decreased 120 minutes after food intake, and 13 biomarkers decreased both 30 and 120 minutes after food intake. Fourteen biomarkers changed 10% or more after food intake, all 120 minutes after food intake: Heat shock 27 kDa protein (10%), Proto-oncogene tyrosine-protein kinase Src (13%), Growth hormone (13%), Carbonic anhydrase 5A, mitochondrial (14%), Carcinoembryonic antigen related cell adhesion molecule 8 (15%), Fatty acid-binding protein, intestinal (16%), Pentraxin-related protein PTX3 (17%), Fibroblast growth factor 21 (18%), C-C motif chemokine 3 (25%), 2,4-dienoyl-CoA reductase, mitochondrial (28%), Gastrotropin (36%), Poly [ADP-ribose] polymerase 1 (37%), Interleukin-6 (42%), and Melusin (52%). Conclusions: The present study shows that food intake affects several different biomarkers analyzed with the Proseek Multiplex CVD II kit, and the effect is at times substantial. Timing of blood sampling in relation to food intake, therefore, appears to be a major concern. Further studies are warranted in older healthy subjects and in patients with various cardiac diseases to determine whether the findings are reproducible.