Background <p>Sleep is essential for maintaining the body’s balance, particularly in regulating the Hypothalamic-pituitary-adrenal (HPA) axis, which controls cortisol, the main stress hormone. Cortisol follows a daily rhythm, but irregular sleep can disrupt this pattern, leading to mood changes, metabolic disorders, and cognitive problems. However, there is limited evidence on effect of sleep duration on cortisol levels among medical students in sub-Saharan Africa. Hence, this study investigated this relationship among selected University’s medical students.</p> Materials and methods <p>This cross-sectional study collected demographic, anthropometric, blood glucose, and saliva cortisol data from 100 healthy Walter Sisulu University medical students aged 18–30; cortisol concentrations were measured once daily (morning) and subsequently analyzed using ELISA kits.</p> Results <p>This study found that sleep duration was not significantly influenced by age, sex, or reported sleep disturbances among medical students. However, a positive and statistically significant correlation was found between sleep duration and salivary cortisol levels (<i>r</i> = 0.2328, <i>p</i> = 0.0198), indicating that students who slept longer exhibited higher cortisol concentrations. Anthropometric variables (BMI, waist, hip, and neck circumference) did not significantly predict cortisol levels, although BMI and hip circumference showed a modest association with blood glucose (R² = 0.093, <i>p</i> ≈ 0.05). In addition, sex showed no significant effect on the sleep-cortisol relationship.</p> Conclusion <p>This study found a positive relationship between sleep duration and cortisol levels, indicating that longer sleep duration alone is not associated with lower cortisol levels in this sample. Cortisol secretion isn’t solely influenced by sleep, highlighting the need for interventions to enhance stress management, sleep quality, and overall well-being among medical students.</p> Clinical trial number <p>Not applicable.</p>