Assessment of Cardiovascular and Stress Biomarkers During Professional Examination Periods in Medical Students: A Prospective Longitudinal Observational Study

ABSTRACT Background: Professional medical examinations induce substantial psychological distress. This prospective longitudinal study evaluated within-participant shifts in cardiac autonomic modulation, resting blood pressure, endocrine markers, and perceived stress between a baseline non-examination period (T1) and an anticipatory pre-examination period (T2). Methods: In this prospective repeated-measures cohort, 50 MBBS students were enrolled and assessed at T1 (6–8 weeks before examinations) and T2 (1–3 days before the first professional examination); 46 students with complete, quality-assured paired data were analysed (aged 20.4 ± 1.2 years; 52.2% female). The primary outcome was 5-minute resting Root Mean Square of Successive Differences (RMSSD). Secondary outcomes included Standard Deviation of NN intervals (SDNN), Heart Rate (HR), blood pressure (SBP, DBP, MAP), salivary cortisol, salivary alpha-amylase 1 (AMY1) protein concentration (ELISA; enzymatic activity was not measured), and Perceived Stress Scale (PSS-10) scores. Paired t-tests and correlation analyses of paired differences (Δ = T2 − T1) were conducted. Results: Of 50 enrolled students, 46 (92%) had complete paired assessments meeting quality criteria and were analysed (2 lost to follow-up, 2 excluded for data quality). Resting RMSSD declined significantly from 42.8 ± 11.3 ms at T1 to 29.4 ± 9.6 ms at T2 (Δ = −13.4 ms, 95% CI: −16.1 to −10.7, p < 0.001, Cohen’s dz = 1.48). Significant increases occurred in resting HR (Δ = +9.2 bpm), SBP (Δ = +6.2 mmHg), DBP (Δ = +4.1 mmHg), salivary cortisol (Δ = +3.12 nmol/L), salivary AMY1 protein (Δ = +14.2 ng/mL), and PSS-10 (Δ = +6.8) (p < 0.001 for all). Within-person ΔRMSSD correlated moderately with ΔPSS-10 (r = −0.42, p = 0.004) but showed no significant correlation with Δcortisol (r = −0.19, p = 0.205). Conclusions: The pre-examination period was associated with reduced RMSSD, higher heart rate and blood pressure, and higher salivary cortisol, salivary AMY1 protein concentration and perceived stress. Changes in physiological and psychological measures were not uniformly correlated within individuals, suggesting domain-specific response patterns. Keywords: Heart Rate Variability, RMSSD, Salivary Cortisol, Salivary AMY1, Perceived Stress Scale, Medical Education, STROBE.