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Introduction. The purpose of this study was to evaluate the application of the Dmax method on heart rate variability (HRV) to estimate the lactate thresholds (LT), during a maximal incremental running test (MIRT). Methods. Nineteen male runners performed two MIRTs, with the initial speed at 8 km·h−1 and increments of 1 km·h−1 every 3 minutes, until exhaustion. Measures of HRV and blood lactate concentrations were obtained, and lactate (LT1 and LT2) and HRV (HRVTDMAX1 and HRVTDMAX2) thresholds were identified. ANOVA with Scheffe’s post hoc test, effect sizes (d), the bias ± 95% limits of agreement (LoA), standard error of the estimate (SEE), Pearson’s (r), and intraclass correlation coefficient (ICC) were calculated to assess validity. Results. No significant differences were observed between HRVTDMAX1 and LT1 when expressed for speed (12.1 ± 1.4 km·h−1 and 11.2 ± 2.1 km·h−1; p=0.55; d = 0.45; r = 0.46; bias ± LoA = 0.8 ± 3.7 km·h−1; SEE = 1.2 km·h−1 (95% CI, 0.9–1.9)). Significant differences were observed between HRVTDMAX2 and LT2 when expressed for speed (12.0 ± 1.2 km·h−1 and 14.1 ± 2.5 km·h−1; p=0.00; d = 1.21; r = 0.48; bias ± LoA = −1.0 ± 1.8 km·h−1; SEE = 1.1 km·h−1 (95% CI, 0.8–1.6)), respectively. Reproducibility values were found for the LT1 (ICC = 0.90; bias ± LoA = −0.7 ± 2.0 km·h−1), LT2 (ICC = 0.97; bias ± LoA = −0.1 ± 1.1 km·h−1), HRVTDMAX1 (ICC = 0.48; bias ± LoA = −0.2 ± 3.4 km·h−1), and HRVTDMAX2 (ICC = 0.30; bias ± LoA = 0.3 ± 3.5 km·h−1). Conclusions. The Dmax method applied over a HRV dataset allowed the identification of LT1 that is close to aerobic threshold, during a MIRT.