Abstract: Ocimum sanctum L. is known for its medicinal properties and also has been considered sacred in India since Vedic times. The aqueous suspension of 70% alcoholic extract of Ocimum sanctum L. was investigated for antifatigue activity. Rats were subjected to Weight-loaded Forced Swim Test (WFST) every alternate day for 2 weeks. O. sanctum at a dosage of 150, 300 and 450 mg kg-1 b.wt. was administrated to rats every day. Swimming time, change in body weight, lipid peroxidation, Lactic Acid (LA), glycogen and blood biochemical parameters namely heamoglobin (Hb%), Blood Urea Nitrogen (BUN) and Creatine Kinase (CK) were evaluated as biomarkers of physical fatigue. The O. sanctum treatment was found to lower malondialdehyde (MDA) and lactic acid levels in liver and muscle tissues compared with control exercise group (p<0.05). Serum biochemical parameters viz., BUN and CK activity were also reduced as compared with control exercise group (p<0.05). The present study thus indicates that out of three studied treatments of O. sanctum (O. sanctum -150, 300 and 450); the O. sanctum at the dose of 300 mg kg-1 b.wt. showed better performance against fatigue when compared with other two treatments. As treatment at 300 mg kg-1 b.wt. facilitated the aerobic glucose metabolism and promote swimming time, suggesting that O. sanctum ameliorates the various impairments associated with physical fatigue.