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This study investigated the acute effect of maximal cycling sprint against difference resistances on intraocular pressure (IOP) depending on participants’ fitness level.In total, 26 physically active collegiate men performed 5 maximal cycling sprints against different resistances in a randomized order, and IOP was measured immediately before and after each sprint. Participants were divided in 2 subgroups (low fit and high fit) according to their maximum power output relative to body weight to assess the influence of fitness level. Two identical testing sessions were performed to assess the repeatability of IOP values.We found that IOP decreases with the lightest resistance (P<0.01), whereas IOP increases with heavier resistances (P<0.01), and it showed a positive linear tendency (r=0.99). Our results suggest that participants’ fitness level seems to influence IOP responses, with a more stable response in the high-fit group. A strong intersession repeatability of IOP values was observed (intraclass correlation coefficient range, 0.82 to 0.98; coefficients of variations range, 1.76% to 6.23%).We conclude that (1) IOP is sensitive to cycling resistance in all-out sprints, with a lowering effect on the lightest resistance and an increasing effect with medium and heavy resistances, (2) high fitness level is beneficial to avoid IOP fluctuations during sprints, and (3) these changes are comparable when measured on 2 different days under the same experimental conditions. A progressive involvement in physical activity may be desirable to avoid IOP peaks during highly demanding physical effort. Future studies are needed to clarify the consequences of exercise in glaucoma patients.