Introduction Nunan et al. (1) found no association between resting Heart Rate Variability (HRV) and the increase in Heart Rate (HR) during a maximal exercise. Hynynen et al. (2), however, have pointed to a Cardiac Autonomic Resource Hypothesis, by which a greater resting vagal tone may allow a further sympathetic activation during a cognitive task (i.e., a higher reduction in vagally-mediated HRV indices); enabling, in turn, a better performance. Therefore, our purpose was to investigate whether resting vagal activity is related or not to sympathetic activation and workload during a 600-m running test. Methods 19 male 800-m runners, belonging to either Senior [n=10; Season Best (SB800-m) 113.75±4.08 s] or Junior category (n=9; SB800-m: 120.92±3.33 s) performed a maximal 600-m run. Heart interbeat intervals (RR) were recorded during 10 min using a Polar RS800 in a seated position before the warm-up and during a 600-m running test. Afterwards, an artifact-free 5-min epoch from resting recording was analyzed using Kubios HRV software. Root-mean-square difference of successive RR intervals (RMSSD) and high-frequency power of RR intervals (HF) were examined as measures of vagal activity, and ratio of low-frequency to high-frequency power (LF/HF) retained as an index of sympathovagal balance (3). Meanwhile, according to Leeper et al. (4), difference between resting and peak HR achieved during the test was employed as a measure of sympathetic activation (ΔHR). Mean velocity during the test was expressed as a percentage of each athlete’s SB800-m (%SB800-m) to obtain a relative measure of workload, inasmuch as the sample consisted of athletes of different age categories. A partial correlation, controlling for resting HR, was conducted to analyze possible relationships between RMSSD, HF, LF/HF, ΔHR and %SB800-m. Previously, HRV measures were logarithmically transformed to allow parametric statistical analysis. Results An inverse significant relationship was found between ΔHR and resting LF/HF (r=-0.53; p<0.05). Furthermore, %SB800-m displayed a significant positive association with resting HF (r=0.48; p<0.05) and a tendency towards significance with RMSSD (r=0.43; p=0.77). Conclusions Our results showed that those athletes with a greater dominance of vagal activity over sympathetic drive (i.e., lower LF/HF) before beginning the training, were capable afterwards of managing a further sympathetic activation during the workout. Moreover, those runners with a greater resting vagal tone (higher HF and RMSSD) achieved a higher relative workload during the 600-m running test. Thereby, current results confirm previous assumptions from Hynynen et al. (2); and as a foremost conclusion, it may be suggested that analysis of HRV prior to high-intensity workouts may provide coaches with a useful information in relation to athletes’ subsequent responses.