(1) Background: Probiotics in the form of nutritional supplements are safe and potentially useful for strategic application among endurance athletes. Bifidobacterium animalis lactis BL-99 (BL-99) was isolated from the intestines of healthy Chinese infants. We combined plasma-targeted metabolomics and fecal metagenomics to explore the effect of 8 weeks of BL-99 supplementation on cross-country skiers’ metabolism and sports performance. (2) Methods: Sixteen national top-level male cross-country skiers were recruited and randomly divided into a placebo group (C) and a BL-99 group (E). The participants took the supplements four times/day (with each of three meals and at 21:00) consistently for 8 weeks. The experiment was conducted in a single-blind randomized fashion. The subject’s dietary intake and total daily energy consumption were recorded. Blood and stool samples were collected before and after the 8-week intervention, and body composition, muscle strength, blood biochemical parameters, plasma-targeted metabolomic data, and fecal metagenomic data were then analyzed. (3) Results: The following changes occurred after 8 weeks of BL-99 supplementation: (a) There was no significant difference in the average total daily energy consumption and body composition between the C and E groups. (b) The VO_2max and 60°/s and 180°/s knee joint extensor strength significantly increased in both the C and E groups. By the eighth week, the VO_2max and 60 s knee-joint extensor strength were significantly higher in the E group than in the C group. (c) The triglyceride levels significantly decreased in both the C and E groups. In addition, the LDL-C levels significantly decreased in the E group. (d) The abundance of Bifidobacterium animalis increased two-fold in the C group and forty-fold in the E group. (e) Plasma-targeted metabolomic analysis showed that, after eight weeks of BL-99 supplementation, the increases in DHA, adrenic acid, linoleic acid, and acetic acid and decreases in glycocholic acid and glycodeoxycholic acid in the E group were significantly higher than those in the C group. (f) Spearman correlation analysis showed that there was a significant positive correlation between Bifidobacterium animalis’ abundance and SCFAs, PUFAs, and bile acids. (g) There was a significant correlation between the most significantly regulated metabolites and indicators related to sports performance and lipid metabolism. (4) Conclusions: Eight weeks of BL-99 supplementation combined with training may help to improve lipid metabolism and sports performance by increasing the abundance of Bifidobacterium, which can promote the generation of short-chain fatty acids and unsaturated fatty acids, and inhibit the synthesis of bile acids.

Introduction: The aim of this study was to combine proteomics and metabolomics to evaluate the immune system of short‐track speed skaters (STSS) before and after a training course. Our research focused on changes in urinary proteins and metabolites that have the potential to serve as indicators for training load.Methods: Urine samples were collected from 21 elite STSS (13 male and 8 female) of the China National Team before and immediately after one training course. First‐beat sports sensor was used to monitor the training load. Proteomic detection was performed using a Thermo UltiMate 3000 ultra high performence chromatography nano liquid chromatograph and an Orbitrap Exploris 480 mass spectrometer. MSstats (R package) was used for the statistical evaluation of significant differences in proteins from the samples. Two filtration criteria (fold change [FC] > 2 and p < 0.05) were used to identify the differential expressed proteins. The Kyoto Encyclopedia of Genes and Genomes enrichment analysis for differential proteins was performed to identify the pathways involved. Nontargeted metabolomic detection was performed using ultra performance liquid chromatography tandem mass spectrometry (UPLC‐MS/MS_) with an ACQUITY 2D UPLC plus Q Exactive (QE) hybrid Quadrupole‐Orbitrap mass spectrometer. Differential metabolites were identified using non‐parametric statistical methods (Wilcox's rank test). Two filtration criteria (FC > 1.2 and p < 0.05) were used to identify differential metabolites. Combined analysis of proteomic and metabolomics were performed on the “Wu Kong” platform. Correlation analysis was performed using Spearman's rank correlation coefficient.Results: (1) The most upregulated proteins were immune‐related proteins, including complement proteins (C9, C4–B, and C9) and immunoglobulins (IgA, IgM, and IgG). The most downregulated proteins were osteopontin (OPN) and CD44 in urine. The correlation analysis showed that the content of OPN and CD44 (the receptor for OPN) in urine were significantly negatively correlated with the upregulated immune‐related proteins. The content of OPN and CD44 is sex‐dependent and negatively correlated with the training load. (2) The most upregulated metabolites included lactate, cortisol, inosine, glutamine, argininosuccinate (the precursor for arginine synthesis), 3‐methyl‐2‐oxobutyrate (the catabolite of valine), 3‐methyl‐2‐oxovalerate (the catabolite of isoleucine), and 4‐methyl‐2‐oxopentanoate (the catabolite of leucine), which is sex‐dependent and negatively correlated with OPN and CD44. (3) The joint analysis revealed five main related pathways, including the immune and innate immune systems. The enriched immune‐related proteins included complements, immunoglobulins, and protein catabolism‐related proteins. The enriched immune‐related metabolites included cAMP, N‐acetylgalactosamine, and glutamate. (4) There is a significant negative correlation between the content of OPN and CD44 in urine and the training load.Conclusion: One training course can lead to the activation of the immune system and a sex‐dependent decrease in the content of OPN and CD44. Training load has a significant and negative correlation with the content of OPN and CD44, suggesting that OPN and CD44 could be potential indicators for training load.

Objectives: We aimed to investigate the effects of 6 weeks of leucine supplementation on athletic performance, central fatigue, and serum metabolome in endurance athletes, and to provide valuable insights into nutritional strategies for endurance athletes. Methods: Twenty cross-country skiers were recruited and randomized into 2 groups: the placebo (PLA) group and the leucine (LEU) group. Subjects were given leucine (8.5 g) + sucrose (14 g) or only sucrose (14 g) supplements twice each day from Monday to Saturday for 6 weeks. Test parameters include body composition, aerobic capacity, isokinetic muscle strength, blood biochemistry, and targeted metabolomics. Results: After intervention, compared to the PLA group (1) the ankle muscle strength (p = 0.01), VO₂max (p = 0.01) and valine in serum (p = 0.03) were increased in the LEU group. (2) Targeted metabolomics results showed that the differential metabolites were enriched in the branched chain amino acids (BCAAs) biosynthesis and degradation. (3) The LEU group had a significant increase in α-ketoisovaleric acid (p = 0.03), which can reduce the continuous decomposition of BCAAs. Conclusions: In conclusion, a six-week intervention of leucine supplementation significantly enhanced ankle muscle strength in endurance athletes, likely through a reduction in BCAA catabolism. Additionally, this combined supplementation strategy demonstrated potential benefits in improving aerobic endurance and may contribute to the attenuation of exercise-induced central fatigue.