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Abstract Prolonged esports play induces cognitive fatigue that is not fully captured by subjective awareness, motivating practical, non-stimulant nutritional strategies supported by objective physiological markers. We here tested whether acute milk protein intake attenuates fatigue-related physiological responses during prolonged esports play and supports subjective state, executive control, and in-game performance. In a randomized, single-blind (assessor-blind), energy-matched controlled crossover study, 15 healthy young adults with esports experience completed two sessions in which they consumed either a milk protein drink or an energy-matched apple juice control before a 3-h virtual soccer task. Physiological measures included pupillometry during gameplay, salivary cortisol, continuous interstitial glucose monitoring, and heart rate. Subjective ratings (VAS) and executive function (flanker task) were assessed across post-ingestion time points, and in-game performance metrics were aggregated within hourly gameplay blocks. Milk protein intake was associated with a coherent pattern of physiological advantages, including larger pupil diameter during gameplay, smoother interstitial glucose dynamics, and lower salivary cortisol, while heart rate showed time-dependent changes without a clear condition effect. These physiological changes co-occurred with higher enjoyment and lower hunger, improved flanker performance, and condition-dependent improvements in in-game performance, most notably higher shot success rate. Additionally, pupil diameter during gameplay was associated with inhibitory-control efficiency on the flanker task. These findings suggest that acute milk protein intake may serve as a practical, non-stimulant nutritional strategy to sustain physiological state and cognitive–behavioral performance during prolonged esports (virtual soccer) play. Highlights – Prolonged esports play models modern digital cognitive activity and cognitive fatigue. – Acute milk protein intake increases pupil diameter during prolonged esports play. – Interstitial glucose dynamics are smoother and salivary cortisol is lower with milk protein. – Enjoyment increases and hunger decreases during 3 h of virtual soccer play. – Executive function and in-game performance improve, most notably shot success rate.