The homeostatic challenge may provide unique opportunities for quantitative assessment of the health-promoting effects of nutritional interventions in healthy individuals. Objective. The present study is aimed at characterizing and validating the use of acute aerobic exercise (AAE) on a treadmill at 60% of VO_2max for 30 min, in assessing the antioxidative and anti-inflammatory effects of a nutritional intervention. In a controlled, randomized, parallel trial of Korean black raspberry (KBR) (n=24/group), fasting blood and urine samples collected before and following the AAE load at either baseline or 4-week follow-up were analyzed for biochemical markers, ¹H-NMR metabolomics, and transcriptomics. The AAE was characterized using the placebo data only, and either the placebo or the treatment data were used in the validation. The AAE load generated a total of 50 correlations of 44 selected markers, based on Pearson’s correlation coefficient analysis of 105 differential markers. Subsequent mapping of selected markers onto the KEGG pathway dataset showed 127 pathways relevant to the AAE load. Of these, 54 pathways involving 18 key targets were annotated to be related to oxidative stress and inflammation. The biochemical responses were amplified with the AAE load as compared to those with no load, whereas, the metabolomic and transcriptomic responses were downgraded. Furthermore, target-pathway network analysis revealed that the AAE load provided more explanations on how KBR exerted antioxidant effects in healthy subjects (29 pathways involving 12 key targets with AAE vs. 12 pathways involving 2 key targets without AAE). This study provides considerable insight into the molecular changes incurred by AAE and furthers our understanding that AAE-induced homeostatic perturbation could magnify oxidative and inflammatory responses, thereby providing a unique opportunity to test functional foods for antioxidant and anti-inflammatory purposes in clinical settings with healthy subjects.