PURPOSE: The present study aimed to identify markers of training stress that were related to the incidence of overreaching in response to overload training in middle distance runners. METHODS: Twenty-four middle distance runners (n=16 M, V̇O2peak: 73.3±4.3 mL·kg·min-1; n=8 F, V̇O2peak: 63.2±3.4 mL·kg·min-1) completed 3 wk of normal training, followed by 3 wk of high-volume training (HVTr; 10% increase in volume each successive week), and a 1-wk taper (TapTr; 55% exponential reduction in training volume from HVTr wk 3). Before, and immediately after each training phase, an incremental treadmill test was performed to measure time to exhaustion (TTE), peak heart rate (HRpeak), HR recovery, peak blood lactate concentration ([La]bpeak) and V̇O2peak. In addition, resting metabolic rate (RMR), body composition, energy intake and resting blood biomarkers of training stress were measured. Runners who had a decreased TTE (>CV) after HVTr were classified as being functionally overreached (FOR), others as acutely fatigued (AF; no decrease performance). Differences between AF and FOR were analysed using mixed-model ANOVAs with pairwise comparisons. RESULTS: Following HVTr, there were significant between group differences in the responses to exhaustive running, whereby FOR (n=12) had a decrease in TTE (-49 ± 14 s), HRpeak (-4 ± 3 BPM; p = 0.02), [La]bpeak (-4.30 ± 1.80 mmol·L-1; p = 0.01) and V̇O2peak (-2.33 ± 2.0 mL·kg·min-1,) and a faster HR recovery (-5 ± 4 BPM; p = 0.03) compared to AF (n=12). There were no between group differences in these variables during submaximal running, nor were there changes in absolute or relative RMR, while only the FOR group increased energy intake during the HVTr. There was no statistically significant change in body composition in either group throughout the study nor were there changes in resting blood biomarkers that reflected inflammation (IL-6, GDF-15 and CRP), metabolism (thyroid hormones), catabolism and anabolism (DHEA, urea, total protein, testosterone, cortisol and GH) or iron regulation (iron, ferritin, and UIBC). CONCLUSIONS: Middle distance runners who were classified as FOR following an overload training period did not have alterations in RMR, resting blood biomarkers or submaximal exercise responses compared to runners who did not have impaired performance.