Approximately 50% of clinicians experience excessive emotional, physical, and mental stress, with repercussions across the entire medical system. Mindfulness exercises may mitigate this excessive stress. Heart rate variability (HRV) is an objective stress measure that can quantify which mindfulness exercises provide the greatest stress reduction. To define the impact of specific mindfulness exercises on HRV, a surrogate for physiologic stress, and the relationship between physiologic (HRV) and subjective stress measured by the State-Trait Anxiety Inventory during a one-day mindfulness workshop. This was a prospective observational pilot study performed at a quaternary children's hospital with diverse subspecialists of pediatric nurses, nurse practitioners, and physicians. Our primary outcome measure was change in HRV from baseline during three mindfulness exercises. The grounding, deep breathing, and body scan exercises all produced statistically significant changes in HRV among our 13 female participants. The body scan exercise produced statistically significant changes in all studied HRV parameters compared with baseline. We observed significant increases in Root Mean Square of Successive Differences between normal heartbeats (p = 0.026), high frequency (p ≤ 0.001), and the parasympathetic nervous system index (p ≤ 0.001) reflecting increased parasympathetic tone (e.g., relaxation), whereas sd 2/sd 1 ratio (p ≤ 0.001) and the stress index (p = 0.004) were decreased reflecting sympathetic withdrawal (e.g., decreased stress). Subjective stress decreased after 1-day mindfulness training (44.6 to 27.2) (p < 0.001). Individuals with the largest decrease in subjective stress also had the most improvement in HRV during the body scan exercise. Clinician stress levels (HRV) improved after participating in grounding, deep breathing, and body scan meditations, which may highlight their importance as stress reduction tools for clinicians. Monitoring of HRV during mindfulness exercises may provide deeper understanding of which specific exercises produce the greatest physiologic stress reduction for individual participants and the trend of these changes over time.