This study aims to analyze the agreement and precision between heart rate variability thresholds (HRVT1/2) with ventilatory and lactate thresholds 1 and 2 (VT1/2 and LT1/2) on a treadmill. Thirty-four male students were recruited. Day 1 consisted of conducting a health survey, anthropometrics, and Cardiopulmonary Exercise Test (CPx). On Day 2, after 48 h, a second incremental test was performed, the Cardiopulmonary Stepwise Exercise Test consisting of 3 min stages (CPxS), to determine VT1/2, LT1/2, and HRVT1/2. One-way repeated-measures ANOVA and effect size (ηp2) were used, followed by Sidak’s post hoc. The Coefficient of Variation (CV) and Typical Error (TE) were applied to verify the precision. Bland Altman and the Intraclass Correlation Coefficient (ICC) were applied to confirm the agreement. HRVT1 showed different values compared to LT1 (lactate, RER, and R-R interval) and VT1 (V̇E, RER, V̇CO2, and HR). No differences were found in threshold 2 (T2) between LT2, VT2, and HRVT2. No difference was found in speed and V̇O2 for T1 and T2. The precision was low to T1 (CV > 12% and TE > 10%) and good to T2 (CV < 12% and TE < 10%). The agreement was good to fair in threshold 1 (VT1, LT1, HRVT1) and excellent to good in T2 (VT1, LT1, HRVT1). HRVT1 is not a valid method (low precision) when using this protocol to estimate LT1 and VT1. However, HRVT2 is a valid and noninvasive method that can estimate LT2 and VT2, showing good agreement and precision in healthy adults.