Lactate threshold (LT) and second ventilatory threshold (VT2) are variables determined during the incremental running test. LT and VT2 are associated with endurance performance [1], [2] and can be used for training prescriptions. However, LT and VT2 measurements require expensive equipment and qualified personnel – which makes their practical application difficult for coaches and athletes. To this concern, the analysis of heart rate variability threshold (HRVT) using a portable cardiac monitor during a maximal incremental test is an alternative and affordable method to estimate the LT and the VT2 [3]. Previous studies indicate that the second HRVT (HRVT2) is strongly associated with LT, and VT2, which could be an alternative low-cost measurement to use during training prescriptions [3], [4], [5]. Peak running velocity (Vpeak) is another low-cost measurement been used for training prescription and it has been associated with running performance [6], and to LT [7], [8]. However, it is not entirely clear whether HRVT2 may be associated to running performance and Vpeak. Understanding these associations would benefit coaches and sports scientists on performance assessments and training prescriptions with more practical and affordable measures. Thus, the aim of this study was to explore the relationships of HRVT2 with Vpeak and 5- and 10-km performance running. Sixteen amateur runners (mean ± SD: age: 34.5 ± 6.0 years; body mass: 71.3 ± 9.0 kg; height: 1.77 ± 0.08 m) took part in this study. Participants were required to visit the laboratory on four separate occasions. In the first session, anthropometric measurements and a maximum incremental test were performed to determine the Vpeak and HRVT. The test started at 6 km h −1, with increments of 1 km h −1 every 3 min and a constant slope of 1%, until voluntary exhaustion. The Vpeak of the incremental test was determined as the velocity of the last complete stage added to the completed fraction of the incomplete stage, determined according to Kuiper's equation [9]. For HRVT determination the time-varying spectral analysis was performed using Fourier transformations on the Kubios HRV software (version 3.4.1) with a sliding window of 64 s, every 20 s. The high frequency (HF) band was extended from 0.15 to 1.8 Hz. The third-order equation was used to model the HF peaks (fHFm). The change in HF spectral density was calculated as the product of this model and HF power (fHFm.HFp), and a logarithmic transformation was used to increase sensitivity to instantaneous change (Ln(fHFm.HFp)) [4], [5]. HRVT2 was determined to be the velocity where occur the last abrupt increase of Ln (fHFm.HFp) [4], [5]. Two independent researchers calculated the thresholds for each subject based on the aforementioned criteria. In case of disagreement, a third researcher was consulted, and the majority value was chosen. In the second session, participants were familiarized with the outdoor track. In the third and fourth sessions, the 5- and 10-km running tests were performed on an outdoor track to determine the running performance. The participants were instructed to finish the test as quickly as possible. Participants were instructed to refrain from any exhaustive or unaccustomed exercise and were instructed to not ingest alcohol or caffeinated beverages for 24 h preceding each test. Each test was performed on different days with a minimum interval of 48 h between tests. All sessions were conducted in the same period of the day, aiming to avoid circadian effects in the measures. The Gaussian distribution was observed through the Shapiro-Wilk test. The associations of HRVT2 with Vpeak and 5- and 10-km performance running were tested using the Pearson product-moment correlation coefficient (r) and adjusted coefficient of determination (R2). Statistical significance was set at P < 0.05. All statistical analyses were performed using the software R v. 3.5.3. The results demonstrated that HRVT2 was highly correlated with the running performance of both running distances (5-km: r = –0.89; 10-km: r = –0.82) and with Vpeak (r = 0.85). In addition, very high associations were found between Vpeak and 5-km running performance (r = –0.94) and 10 km running performance (r = 0.85) (Fig. 1).