The purpose was to evaluate the effects of Mindfulness in Motion (MIM), a Mindfulness Based Intervention, on respiration rate, heart rate, and heart rate variability (HRV). Healthcare providers wore chest strap electrocardiography-based (ECG) devices during MIM sessions (n = 84). Data were collected during the following mindfulness-based segments: 5-minute quiet sitting to reflect on the weekly theme that ended with a 30 second mindful breath count (Baseline-MIM), 15-minute Education video, 20-minute Discussion with peers, 15-minute Practice, and lastly a 5-minute reflection ending with a 30 second breath count (Post-MIM). Metrics from ECG included: mean heart rate, root-mean square of successive differences between R-R intervals (rMSSD), standard deviation of R-R intervals (SDNN), absolute power for low frequency (LF, 0.04–0.15 Hz), and high frequency (HF, 0.15–0.4 Hz) bands, Total Power across entire frequency range (< 0.4 Hz), LF:HF Ratio, and respiration rate. According to mixed effect models there were main effects of MIM Session Segments (p < 0.05) across all metrics. Heart rate was elevated during Practice (4.96 ± 0.91, p < 0.001, ES = 0.383), while rMSSD was not significantly different at any timepoint (p > 0.05). Compared to Baseline-MIM, respiration rates were significantly slower in Practice (1.98 ± 0.35 bpm) and Post-MIM (2.03 ± 0.35 bpm, p < 0.001), while SDNN, LF, and LF:HF were significantly increased. During practice, HF was decreased, while Total Power was increased during Post-MIM. Acute improvements in HRV, likely from acute decreases in respiration rates, occurred while healthcare providers completed a mindfulness intervention (MIM) during work hours.