Previous studies have examined the effects of n-3 fatty acid intake in supplement form or fish oil capsules, but there are few studies based on other foods. Perilla oil is a traditional Japanese seed oil rich in n-3 fatty acids. This randomised trial aimed to determine the appropriate n-3 fatty acid dose through consumption of perilla oil, which improves gut function and microbiota in trained athletes, and the amount of fat fuel required to provide energy to athletes involved in high-intensity training to improve athletic performance. Thirty-six female athletes training six times per week were randomly assigned to three groups according to perilla oil intake: 9 g/d (high oil intake (HOI)), 3 g/d (low oil intake (LOI)) and placebo-supplementation (PLA) groups. The HOI and LOI groups had perilla oil-containing jelly and the PLA group had placebo jelly for 8 weeks. Gut microbiota, constipation score and urinary biochemical index were measured pre- and post-intervention. The spoilage bacteria, Proteobacteria, significantly decreased (P = 0·036, d = 0·53), whereas Butyrate-producing bacteria, Lachnospiraceae, significantly increased (P = 0·007, d = 1·2) in the HOI group. Urinary indoxyl sulphate significantly decreased in the HOI group only (P = 0·010, d = 0·82). Changes in the constipation score were significantly lower in the HOI group (P = 0·020) and even lower in the LOI group (P = 0·073) than in the PLA group; there were significant differences between groups (P = 0·035). Therefore, perilla oil intake may improve gut function and microbiota in athletes, with higher doses resulting in further improvement.