Background: Ingestion of combination of carbohydrates (CHO) is beneficial for exercise performance. Honey is a natural readily available source of CHO mixture which possesses anti-oxidant and immune boosting properties, so it might enhance endurance performance by maintaining blood glucose level during exercise and reducing exercise induced oxidative stress. The present study was conducted to investigate the efficacy of acute supplementation of honey on endurance performance in healthy male individuals. Methodology: In this PL-controlled double-blind randomized study, 12 healthy males (20-25 years) were supplemented with Honey 30 g or Honey 60 g before they performed the treadmill running at a speed that elicited 80% of HRmax after an overnight fast. Pre–exercise HR, body temperature and RPE were also recorded. Blood samples were drawn before supplementation and at the time of exhaustion for analysis of glucose, insulin, lactic acid, free fatty acid, superoxide dismutase, lipid peroxidation, catalase and total thiol. Data were analyzed by two way RM ANOVA followed by post hoc test. Results: Endurance time showed significant (p<0.05) difference between the PL and honey trials as well as between H30 and H60 trials. Changes in working heart rate (HR) were significantly (p<0.05) different over time in the same experimental trial compared to the respective resting value. Working HR had no significant difference between H30 and H60 trials, except at 90th min and at the time of exhaustion. H30 trial showed significant (p<0.05) difference in working HR in comparison to PL trial at 0th, 20th, 40th, 50th, 60th and 80th min. Working HR of H60 trial were also significantly (p<0.05) different in comparison to PL trial. RPE was significantly (p<0.05) different at the time of exhaustion in comparison to the 10th min value of the corresponding trial. Values of lactate, glucose, insulin, free fatty acid, lipid peroxidase, total thiol, catalase and superoxide dismutase depicted significant difference not only between H30 and H60 trials but also between PL trial and two different honey trials. Interpretation and conclusion: Acute honey supplementation has significant beneficial effect on endurance performance and biochemical blood parameters. So it can be used as a natural ergogenic aid to boost exercise performance. © 2020, Association of Physiologists and Pharmacologists of India. All rights reserved.