Probiotic Lactobacillus kefiranofaciens K6 attenuates physiological and behavioral alterations and modulates the gut microbiota in a mouse model of overtraining syndrome

Abstract

Exercise is widely recognized for its substantial health benefits in promoting well-being and preventing disease across diverse populations; however, inappropriate training intensity combined with insufficient recovery can lead to overtraining syndrome (OTS), characterized by sustained performance decline, physiological maladaptation, and psychological disturbances. Probiotics have emerged as a promising nutritional strategy for modulating host metabolism and the gut microbiota, yet effective interventions for OTS remain limited. In the present study, a well-established OTS-like mouse model induced by a progressive treadmill protocol was used to evaluate the effects of Lactobacillus kefiranofaciens K6 supplementation on performance decline, physiological maladaptation, psychological disturbances, and gut microbiota dysbiosis. Mice were randomly assigned to Sedentary, Exercise, OTS, and OTS-K6 groups and received the designated interventions for 10 weeks. K6 supplementation was associated with attenuation of OTS-induced reductions in body weight, appetite, body composition, endurance performance, glucose tolerance, and tissue glycogen content, as well as improvements in peripheral fatigue biomarkers, including muscle injury markers (CK and LDH) and energy metabolism indicators (glucose, lactate, and ammonia). In addition, K6 administration was associated with improved intestinal barrier-related gene expression (Claudin-1, ZO-1, and ZO-2), reduced tissue and systemic inflammatory responses (TNF-α, IL-6, neutrophil counts, PLR, and NLR), and support of immune function as indicated by lymphocyte levels. K6 supplementation also alleviated OTS-induced anxiety-like behaviors. Furthermore, gut microbiota composition was altered by exercise, OTS induction, and K6 supplementation, with K6 modulating OTS-related dysbiosis by reducing Heminiphilus faecis and increasing Paramuribaculum intestinale. Moreover, changes in Duncaniella dubosii and P. intestinale may also provide potential benefits in the Exercise group. Excessive training stress in this experimental OTS-like mouse model was associated with multiple metabolic, inflammatory, behavioral, and microbiota-related disturbances, whereas K6 supplementation was associated with attenuation of several OTS-related alterations. These findings suggest that microbiota-targeted nutritional strategies may support physiological resilience under excessive training stress; however, the results should be interpreted as associative and hypothesis-generating, as mechanistic relationships were not directly examined.