Objective To quantify physician stress levels when performing lumbar puncture (LP) and explore operator stress effect on patient outcomes. Methods This was a cross-sectional, multicenter study. Novices, intermediates, and experts in performing LP were recruited from 4 departments of neurology and emergency medicine. Stress was measured before and during performance of the LP using cognitive appraisal (CA), State-Trait Anxiety Inventory-Short (STAI-S) questionnaire, and the heart rate variability measure low frequency/high frequency index (LF/HF ratio). Patient-related outcomes were pain, confidence in the operator, and postdural puncture headache (PDPH). Results Forty-six physicians were included in the study: 22 novices, 12 intermediates, and 12 experts. Novices had the highest stress level and experts the lowest measured by cognitive appraisal and STAI-S before and during LP performance (p < 0.001 for all comparisons). Novices had the highest sympathetic tonus indicated by the highest LF/HF ratio before (p = 0.004) and during (p = 0.056) LP performance. Physician stress level was not significantly related to patients' pain. However, there was a significant relationship between STAI-S during the procedure and patient confidence in the operator (regression coefficient = −0.034, p = 0.008). High physician heart rate during the procedure significantly increased the odds of PDPH (odds ratio = 1.17, p = 0.036). Conclusion Novice stress levels were high before and during performance of LP. Stress was significantly related to patient confidence in the operator and risk of PDPH. Simulation-based training should be considered to reduce novice residents' stress levels and increase patient safety.