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Chronic stress, common in university students and particularly pronounced in women, negatively impacts health. Heart Rate Variability (HRV), an autonomic balance measure, is influenced by stress, physical activity (PA), and the menstrual cycle. This study examined HRV and perceived stress differences in healthy-weight female university students by PA level. Fifty-two female university students (18-30 years, BMI 18.5-24.9 kg/m<sup>2</sup>) were recruited. To minimise hormonal variability, assessments for naturally menstruating participants were conducted during the early follicular phase, confirmed through a cycle-tracking application and luteinizing hormone (LH) tests. For participants using hormonal contraceptives, measurements were scheduled during the first week of a new contraceptive cycle. PA was assessed (IPAQ-SF) and categorised as low-moderate or vigorous. HRV was recorded using a Polar H10 over three days for reliability (SDNN, LF, HF, LF/HF ratio) were analysed. Perceived stress used the Perceived Stress Questionnaire (PSQ). Group differences and associations were then analysed, controlling for total PA (METs). Forty-two participants completed the study. Vigorous PA was associated with higher SDNN (p = 0.099, d = -0.620) and lower LF (p = 0.045, d = -0.761) versus the low-moderate PA group, indicating more efficient cardiac autonomic modulation. METs significantly influenced SDNN, HF, LF, and pNN50. However, no significant differences were found in LF/HF ratio (p = 0.990) or perceived stress (p = 0.249) between groups, nor were significant correlations observed between PSQ scores and any HRV indices. Higher PA levels correlate with a more favourable autonomic profile (increased parasympathetic activity, reduced sympathetic drive at rest, unaltered LF/HF balance). The lack of association between PA/perceived stress and perceived stress/HRV suggests a potential dissociation between subjective stress perception and objective autonomic activity in this population.