BACKGROUND: Surgical training and practice is stressful, but adaptive changes in the stress circuitry (e.g. perceptual, physiological, hormonal, neural) could support skill development. This work examined skill acquisition and stress adaptations in novice surgeons during laparoscopic surgery (LS) training and detraining. METHODS: Twelve medical students were assessed for skill performance after 2 h (BASE), 5 h (MID) and 8 h (POST) of LS training in weeks 1-3, and then after 4 weeks of no training (RETEST). The stress outcomes included state anxiety, perceived stress and workload, heart rate (HR), heart rate variability (HRV), and salivary testosterone and cortisol concentrations. Functional near-infrared spectroscopy was used to assess cortical oxygenation change, as a marker of prefrontal cortex (PFC) activity. RESULTS: Skill performance improved in every session from BASE (p < 0.01), with corresponding decreases in state anxiety, stress, workload, low- and high-frequency HRV in the MID, POST and/or RETEST sessions (p < 0.05). Left and right PFC were symmetrically activated within each testing session (p < 0.01). The stress and workload measures predicted skill performance and changes over time (p < 0.05), with state anxiety, mean HR and the HRV measures also showing some predictive potential (p < 0.10). CONCLUSIONS: A 3-week LS training programme promoted stress-related adaptations likely to directly, or indirectly, support the acquisition of new surgical skills, and many outcomes were retained after a 4-week period without further LS training. These results have implications for medical training and education (e.g. distributed training for skill development and maintenance, stress resource and management training) and highlighted possible areas for new research (e.g. longitudinal stress and skill profiling).