PUSHED TO THE LIMIT: DISTINCT EV AND BEHAVIORAL SIGNATURES OF EXERCISE-INDUCED STRESS IN MICE

Acute physical exercise (PE) is used to study stress‑related adaptations, but the mechanisms driving intensity‑dependent responses remain unclear. We investigated how moderate and intense PE affect anxiety‑like behaviors and serum extracellular vesicle (EV)-associated proteomic signatures in mice. Young-adult male C57BL/6 mice were allocated to sedentary (SED), moderate (PEM; 14 m/min), or intense exercise (PEI; 18 m/min). After treadmill habituation, animals completed a single 30‑minute running session. Behavioral testing (Open Field Test, Elevated Plus Maze, Splash Test) was used to assess anxiety-like responses, and was performed 15 minutes post‑exercise across three weeks. A diazepam-treated group served as an anxiolytic control. One week later, animals were euthanized for blood collection. Serum EVs were analyzed by high‑resolution mass spectrometry for proteomic analysis. ANOVA was used for group comparisons (p<0.05). Acute PE produced intensity‑dependent behavioral differences: PEI mice showed fewer open‑arm entries, whereas PEM animals spent less time in open arms and exhibited increased grooming. EV proteomics revealed molecular adaptations, with both PEM and PEI showing modulation of mitochondrial and metabolic pathways relative to SED mice, suggesting systemic bioenergetic stress. Direct PEM–PEI comparisons highlighted differential regulation of inflammatory and lipid‑related processes, with predicted upstream regulators including HIF1A, NR4A1, and FST. Overall, a single bout of forced exercise elicits intensity‑dependent behavioral and EV‑based molecular changes, implicating mitochondrial, metabolic, and inflammatory pathways as potential mediators of exercise‑induced stress.