BACKGROUND: Skill competency assessments induce stress and anxiety and may affect nursing student performance. Little is known about stress and perceived anxiety levels and their relationship in the mock skill competency assessment. METHODS: A cross-sectional study was conducted to examine the stress levels (as assessed by heart rate variability, HRV) and perceived anxiety before, during and after the mock skill competency assessment, and to explore their relationships to performance in a total of ninety first-year undergraduate nursing students. RESULTS: The HRV decreased significantly during the assessment and increased significantly 10 min after the assessment (p < 0.01). Higher performers showed significantly lower HRV during and after the assessment (p < 0.01). The assessment score was negatively correlated with HRV during and after the assessment (p < 0.05). CONCLUSIONS: Considering assessment-related stress and anxiety through a mock assessment prior to the actual skill assessment provides implications for future nursing education.