OBJECTIVE: The aim of this study was to investigate the changes in 24-h heart rate variability and aerobic fitness, and their associations, in female soccer players during the preseason period. METHODS: Sixteen players were assessed (24-h HRV and Yo-Yo Intermittent Recovery Test, level 1 [YYIR1]) before and after 4 weeks of preseason. The relationship between R-R24h length and high-frequency oscillations (HF24h) was analyzed by a quadratic regression model (revealing or not saturation of vagal activity) assessed 48-h before (PRE-preseason) and 48-h after (POST-preseason) the preseason period. Additionally, the mean HF24h was calculated from the linear portion of the R-R interval versus the HF24h regression curve (HF index). The average of the corresponding R-R24h values was defined as the R-R index. RESULTS: In PRE-preseason, seven players had a saturated HF24h, while in POST-preseason, five new cases of saturated HF24h were observed. The mean R-R24h, HF24h, R-R index, and HF index lengths significantly increased after preseason (p < 0.001). Significant differences were found in YYIR1 PRE- compared with POST-preseason (930 +/- 286 m [individual range: 400-1240 m] versus 1265 +/- 252 m [640-1640 m], respectively; p < 0.001). Additionally, the relative changes in HF24h and HF index were largely correlated with improvements in the distance covered during the YYIR1 (r = 0.68 and r = 0.56; respectively). CONCLUSION: Enhanced vagal activity after 4-week preseason period of soccer training increased the occurrence of vagal saturation in high-level female soccer players. Additionally, the increases in HF24h and HF index were significantly correlated with aerobic fitness change.