Abstract: Background　Zusanli (ST36) is one of the most commonly used acupoints in acupuncture and has shown good therapeutic effects on cardiovascular and other systemic diseases. However, the autonomic nervous system modulation pattern of deep needling at ST36 still lacks objective quantitative evidence. Objective　To observe the effects of deep needling (near the blood vessel) at Zusanli on heart rate variability (HRV), blood pressure variability (BPV), cardiac output (CO), and baroreflex sensitivity (BRS) in healthy subjects, thereby elucidating the regulatory effect of acupuncture on cardiovascular autonomic nerves and providing objective evidence for the acupuncture theory of "regulating Qi and blood". Methods　Healthy volunteers were recruited at Chinese PLA General Hospital from October to December 2025 and randomly divided into a deep needling group and a superficial needling group. ST36 was used in both groups. In the deep needling group, a filiform needle was inserted perpendicularly about 30 mm under ultrasound guidance to reach the branches of the anterior tibial artery, followed by lifting-thrusting manipulation for 150 seconds before withdrawal. In the superficial needling group, the needle was inserted about 10 mm without reaching the blood vessel and without any manipulation. Electrocardiogram and continuous blood pressure signals were simultaneously collected for 5 minutes at five time points: before needling (T1), during needling (T2), immediately after needling (T3), 30 min (T4) and 120 min (T5) after needling. HRV, BPV, and CO were analyzed, and BRS was analyzed using transfer function analysis. Results　A total of 68 subjects were enrolled, including 34 in the deep needling group (15 males, 19 females, mean age 26.2±3.2 years) and 34 in the superficial needling group (16 males, 18 females, mean age 25.5±3.3 years). There were no statistically significant differences in age or gender between the two groups (P＞0.05). In terms of HRV, the deep acupuncture group showed significantly higher natural logarithm of the root mean square of successive RR interval differences (lnRMSSD) at T2, T3, and T4 compared with T1 (all P＜0.01), while no significant difference was found between T5 and T1 (P＞0.05); the shallow acupuncture group exhibited no significant differences in lnRMSSD at any time points relative to T1 (all P＞0.05), whereas between-group comparison revealed that ΔT2 (change from T1 to T2) in lnRMSSD was significantly greater in the deep acupuncture group than in the shallow acupuncture group (0.35±0.31 vs 0.13±0.32, P＜0.01). Regarding BPV, the deep acupuncture group demonstrated significantly increased systolic blood pressure standard deviation (SBP-SD) and coefficient of variation (SBP-CV) at T2 compared with T1 (both P＜0.01), with no other time points differing significantly from T1 (all P＞0.05); the shallow acupuncture group showed no significant changes in any BPV parameter across all time points relative to T1 (all P＞0.05), yet between-group analysis indicated that at T2, SBP-SD and SBP-CV in the deep acupuncture group were significantly higher than those in the shallow acupuncture group SBP-SD: (6.5±2.6) mmHg vs (4.9±1.4) mmHg; SBP-CV: 0.057±0.021 vs 0.042±0.011, both P＜0.01. For cardiac output (CO), no significant interaction effect was observed (P＞0.05), although a significant time effect was present (P＜0.05), with post hoc pairwise comparisons showing that CO at T2, T3, and T4 was significantly lower than at T1 (P＜0.05 or P＜0.01). Concerning baroreflex sensitivity (BRS), the deep acupuncture group had significantly elevated BRS gain at T3 compared with T1 (P＜0.05), whereas the shallow acupuncture group exhibited no significant differences in BRS gain across any time points relative to T1 (all P＞0.05); between-group comparison demonstrated that BRS gain at T3 was significantly higher in the deep acupuncture group than in the shallow acupuncture group (10.66 6.81, 17.76 ms/mmHg vs 8.37 7.03, 10.50 ms/mmHg, P＜0.05). Conclusion　Deep needling at Zusanli regulates autonomic nerves for both the heart and blood vessels. For blood vessels, it mainly manifests a sympathetic effect; for the heart, it mainly manifests a parasympathetic effect. This study may provide a preliminary clinical reference for the treatment of cardiovascular diseases such as sinus tachycardia, cardiac neurosis, and orthostatic hypotension.