ABSTRACT Alternate day fasting (ADF) is beneficial in delaying aging and associated morbidities by reducing oxidative stress and inflammation. Hesperidin is a flavonoid antioxidant known for its antioxidant and anti-inflammatory properties. In this study, we aimed to evaluate the synergistic role of hesperidin supplementation and ADF regimen in Wistar rats on biomarkers of aging and inflammation. Middle-aged male Wistar rats (12–15 months) were divided into four groups (n = 6): Group I. Control (given 1.0 mL of carboxymethylcellulose orally); Group II. ADF (chow diet given on alternate days); Group III. Hesperidin (100 mg/kg BW orally); Group IV. ADF+ Hes (ADF regimen and hesperidin dupplemented) for three weeks. We measured clinical parameters and some crucial biomarkers of oxidative stress like FRAP, GSH, PMRS, MDA, PCO, AOPP, NO, insulin, adiponectin and inflammatory cytokines (TNF-α. IL-6, CRP and COX). A significant increase (p < 0.05) in insulin, FRAP and GSH, and a significant decrease (p < 0.05) in MDA and NO were observed in the ADF+Hes group. IL-6, CRP and COX were also significantly reduced (p < 0.05) suggesting hesperidin-induced benefits. We conclude that hesperidin intake during ADF may serve as a crucial purpose in improving signature biomarkers of health and metabolism.