PURPOSE: Implementation of gluten-free diets among nonceliac athletes has rapidly increased in recent years because of perceived ergogenic and health benefits. The aim of this study was to investigate the effects of a gluten-free diet (GFD) on exercise performance, gastrointestinal (GI) symptoms, perceived well-being, intestinal injury, and inflammatory responses in nonceliac athletes. METHODS: Thirteen competitive endurance cyclists (8 males, 5 females) with no positive clinical screening for celiac disease or history of irritable bowel syndrome (mean ± SD; age, 32 ± 7 yr; weight, 71.1 ± 13.4 kg; height, 177.0 ± 11.8 cm, VO2max 59.1 ± 8.0 mL·kg⁻¹·min⁻¹) were allocated to a 7-d gluten-containing diet (GCD) or GFD separated by a 10-d washout in a controlled, randomized, double-blind, crossover study. Cyclists ate a GFD alongside either gluten-containing or gluten-free food bars (16 g wheat gluten per day) while habitual training and nutrition behaviors were controlled. During each diet, cyclists completed the Daily Analysis of Life Demand for Athletes (DALDA) and GI questionnaires (postexercise and daily). On day 7, cyclists completed a submaximal steady-state (SS) 45-min ride at 70% Wmax followed by a 15-min time trial (TT). Blood samples were taken preexercise, post-SS, and post-TT to determine intestinal fatty acid binding protein (IFABP) and inflammatory markers (cytokine responses: interleukin [IL] 1β, IL-6, IL-8, IL-10, IL-15, tumor necrosis factor α). Mixed effects logistic regression was used to analyze data. RESULTS: TT performance was not significantly different (P = 0.37) between the GCD (245.4 ± 53.4 kJ) and GFD (245.0 ± 54.6 kJ). GI symptoms during exercise, daily, and DALDA responses were similar for each diet (P > 0.11). There were no significant differences in IFABP (P = 0.69) or cytokine (P > 0.13) responses. CONCLUSIONS: A short-term GFD had no overall effect on performance, GI symptoms, well-being, and a select indicator of intestinal injury or inflammatory markers in nonceliac endurance athletes.