The aim of this study was to investigate whether the use of handrail support during maximal exercise treadmill testing (ETT) would interfere in cardiac autonomic modulation kinetics when compared to not using handrail support. The hypothesis of overestimation in cardiac autonomic dynamics when the ETT is performed using handrail was tested. Thirty-five undergraduates (21.08 ± 2.98 years old) of both sexes, volunteered to undertake two ETT under the Ellestad protocol, in non-consecutive days. The first test (T1) was performed with handrail support and, after 7 days, the second test was performed (T2) without the support. Autonomic function was measured by heart rate variability (HRV) during both tests and resting. Estimated value of peak oxygen uptake (VO2) was 22.4% (p < 0.0001) higher in T1 when compared to T2. Overall, parasympathetic pathway was deactivated earlier in T2 than in T1, with NNxx measures variating in T1 from 10.74 ± 14.59 (ms) and in T2 from 3.48 ± 3.79 (ms). In stage two, mean values of HF in T2 corresponded to 32% of values in T1. Stage three presented a difference of 60% (p < 0.014) in LF between means reached in T1 and T2. Lastly, the association of LF and VO2 persisted longer in T1 stages than in T2 and was verified in early stages (S2 and S3) of both ETTs. Our findings suggest that parasympathetic influences on HR were slightly prolonged during ETT when subjects hold onto the treadmill.