Introduction: The purpose of this study was to characterize longitudinal patterns associated with non-functional overreaching (NFOR) in NCAA Division I American football athletes across a competitive season using salivary biomarkers, neuromuscular performance measures, and recovery metrics, with exploratory analyses by team role. Methods: Thirty-nine male athletes (age 20.4 ± 1.75 years) were monitored across a 15-week competitive season. Saliva samples collected pre-, mid-, and post-season assessed cortisol, testosterone, interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-a). Neuromuscular performance was evaluated using countermovement jump metrics (jump height, normalized peak power and force, reactive strength index-modified [RSImod]) and PlayerLoad, while recovery was tracked via twice-weekly self-reports of sleep quality and muscle soreness. Associations among biomarkers, recovery, and performance were examined by starting status and position group; statistical significance was set at p = 0.05. Results: Distinct role-dependent patterns emerged. Among starters, higher testosterone-to-cortisol ratios were associated with lower soreness and better sleep quality, while higher IL-6/cortisol ratios were associated with higher RSImod values. Non-starters showed patterns consistent with greater physiological strain, with higher IL-6/cortisol ratios associated with poorer recovery despite lower external loads. Mid-season declines in countermovement jump performance coincided with biomarker changes suggestive of accumulated fatigue. Discussion: Overall, both starters and non-starters exhibited patterns consistent with NFOR, though physiological signatures differed by role. Salivary biomarker ratios, particularly IL-6/cortisol, alongside neuromuscular measures such as RSImod may provide useful, non-invasive indicators of fatigue-related stress and support role-specific recovery strategies in applied sport settings.
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