There is a strong demand for diagnostic tools to identify athletes in various training states. PURPOSE: To determine if a cluster of proteins could be identified through proteomics procedures that are linked to functional overreaching (FOR) in male endurance athletes. METHODS: Participants (N=10, age 38.3±3.4 y, VO2max 41.3±1.7 ml.kg-1.min-1) served as their own controls and in random, counterbalanced order either ran/cycled 2.5 h (70.0±3.7% VO2max, 79.6±6.3% HRmax) three days in a row (FOR) or sat in the lab (rest) (separated by three weeks) (7:00 - 9:30 am, overnight fasted state). Participants provided fingerprick samples for dried blood spot samples (DBS) pre- and post-exercise/rest each of the three days, and then at 7:00 am during two additional recovery days. Participants also completed the Training Distress Scale (TDS) (19-items) at 7:00 am each of the five mornings during each trial (FOR and rest). Proteins were solubilized from DBS, digested into peptides and measured with nanoLC-MS in data independent acquisition mode (Q-Exactive, Thermo Fisher Scientific, Waltham, MA). The RAW MS data files were processed using SpectronautTM software (Biognosys, Schlieren, Switzerland). Following data independent acquisition (DIA method), 594 proteins were identified and quantified. Proteins were considered for the FOR cluster if they were elevated during one of the two recovery days but not more than one of the exercise days (compared to rest). The Generalized Estimating Equation (GEE) was used to identify proteins linked to FOR (between trial contrasts, P≤0.05 for proteins with CV<15%, P≤0.01 with CV>15%). RESULTS: TDS scores differed between FOR and rest trials, peaking on the first recovery day (9.8±3.8, 3.5±2.6, respectively, P=0.029). A total of 13 proteins was linked to FOR and of these, 11 were related to the immune system, and two to exercise-induced physiological responses. Immune-related proteins included those associated primarily with the acute phase response, complement activation, and granulocyte function. CONCLUSIONS: This study utilized targeted, DIA proteomics procedures to identify a cluster of 13 proteins linked to FOR (7.5 h of high intensity exercise over three days), and 85% of the proteins were related to immune system activation during the 2-day recovery period.