Compared to sedentary people, athletes exhibit a much more abundant and diverse composition of gut bacteria. Hence the relationship between gut microbes and energy usage and exercise performance has attracted much attention in recent years. Probiotics and prebiotic-related products have demonstrated the potential to enhance metabolic pathways and influence energy levels, energy consumption and exercise performance. And previous studies demonstrated positive effects on exercise endurance associated with the consumption of kefir, a fermented dairy product containing Lactobacilli species as part of the microbial symbiosis. This study investigated whether kefir can promote changes in the gut microbiota, improve exercise endurance performance, and influences fatigue during and after exercise. The study enrolled sixteen, untrained 20–30-year-old for a double-blind crossover design study, supplementing with SYNKEFIR™ for 28 days whilst observing changes in metabolic markers, body composition, exercise endurance and faecal gut bacteria. In summary, supplementation with SYNKEFIR™ significantly improved exercise performance and reduced the production of lactic acid after exercise. In addition, kefir supplementation seemed to reduce fatigue and accelerated the recovery from fatigue after exercise, with a marked reduction in lactic acid production after exercise. Though kefir supplementation had no significant effect on other post-exercise fatigue biochemical indicators nor did it induce notable changes in gut bacteria composition. As SYNKEFIR™ is a starter culture isolated from traditional kefir it could be expected that other traditional kefir products would have similar effects. Kefir as a food product is suited to a wide range of people, and it could be considered part of a healthy diet plan for untrained individuals wishing to support their exercise performance.