Metabolomic profiling of extracellular vesicles reveals enhanced oxidative stress and energy metabolism during intense military training: an exploratory study

Extracellular vesicles (EVs) are small, membrane-bound vesicles that transfer biological content through the extracellular environment. The role of EVs in energy metabolism has primarily focused on EV proteins and microRNAs, with less attention on the metabolic content of EVs. This exploratory study assessed changes in the EV metabolome in response to an arduous, 16-day military training exercise. Forty male soldiers (21 ± 2 yr, 24.8 ± 2.7 kg/m²) provided blood from which circulating EVs were isolated and completed assessments of body composition and lower body power on <i>days 1</i> (PRE) and <i>16</i> (POST) of a mountain training exercise (MTX). Total daily energy expenditure during the MTX was 4,187 ± 519 kcal·day^-1. Fat mass (POST-PRE [95% confidence interval]: -0.9 [-1.3, -0.6] kg), lean body mass (-1.6 [-2.0, -1.2] kg), body fat percentage (-0.7 [-1.1, -0.3]%), and lower body power (-133 [-204, -63] W) decreased from PRE to POST (<i>P</i> < 0.05). Global metabolite profiling identified 81 metabolites from lipid (81%), energy (5%), cofactor and vitamin (5%), xenobiotic (4%), carbohydrate (2%), amino acid (1%), and nucleotide (1%) pathways in serum-derived EVs. After adjusting for EV concentration, 11 metabolites were different from PRE to POST (<i>P</i> < 0.05, <i>Q</i> < 0.20), with the largest increases in the oxidative stress-associated metabolites 5-oxoproline and benzoate. Changes in lean body mass were positively associated with changes in the energy metabolites citrate (ρ = 0.361, <i>P</i> = 0.022) and phosphate (ρ = 0.369, <i>P</i> = 0.019). Findings suggest that EV metabolites change in response to physiological stress and reflect increased oxidative stress, energy metabolism, and fatty acid metabolism, which may provide early indicators of stress adaptations relevant for optimizing training and sustaining military performance.<b>NEW & NOTEWORTHY</b> EV metabolites change in response to periods of increased metabolic demand, reflecting increased oxidative stress, energy metabolism, and fatty acid metabolism, and may be associated with changes in lean body mass. This exploratory study adds to the limited existing literature by highlighting the potential of EV-derived metabolites to provide insight into metabolic responses and their contribution to stress-induced metabolic adaptations.