Fully restoring autonomic nervous system (ANS) function is paramount for peak sports performance. Training programs failing to provide sufficient recovery, especially during the in-season, may negatively affect performance. This study aimed to evaluate the influence of the physiological workload of collegiate football training on ANS recovery and function during the in-season. Football athletes recruited from a D1 college in the southeastern US were prospectively followed during their 13-week "in-season". Athletes wore armband monitors equipped with ECG and inertial movement capabilities that measured exercise cardiac load (ECL; total heartbeats) and maximum running speed during and baseline heart rate (HR), HR variability (HRV) 24 h post-training. These metrics represented physiological load (ECL = HR.Duration), ANS function, and recovery, respectively. Linear regression models evaluated the associations between ECL, baseline HR, HRV, and maximum running speed. Athletes (n = 30) were 20.2 +/- 1.5 years, mostly non-Hispanic Black (80.0%). Negative associations were observed between acute and cumulative exposures of ECLs and running speed (beta = -0.11 +/- 0.00, p < 0.0000 and beta = -0.15 +/- 0.04, p < 0.0000, respectively). Similarly, negative associations were found between baseline HR and running speed (beta = -0.45 +/- 0.12, 95% CI: -0.70, -0.19; p = 0.001). HRV metrics were positively associated with running speed: (SDNN: beta = 0.32 +/- 0.09, p < 0.03 and rMSSD: beta = 0.35 +/- 0.11, p < 0.02). Our study demonstrated that exposure to high ECLs, both acutely and cumulatively, may negatively influence maximum running speed, which may manifest in a deteriorating ANS. Further research should continue identifying optimal training: recovery ratios during off-, pre-, and in-season phases.