Detection of Functional Overreaching in Endurance Athletes Using Proteomics

Abstract

No reliable biomarkers exist to identify athletes in various training states including functional overreaching (FOR), non-functional overreaching (NFOR), and overtraining syndrome (OTS). Participants (N = 10, age 38.3 ± 3.4 years) served as their own controls and in random, counterbalanced order either ran/cycled 2.5 h (70.0 ± 3.7% VO_2max) 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, and then during two recovery days. DBS proteins were measured with nanoLC-MS in data-independent acquisition (DIA) mode, and 593 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. A total of 13 proteins was linked to FOR and most were associated with the acute phase response and innate immune system activation. This study used a system-wide proteomics approach to define a targeted panel of blood proteins related to FOR that could form the basis of future NFOR- and OTS-based studies.

Keywords: acute phase response; blood proteins; complement; exercise; granulocytes.

Figure 1

Research design with study participants (N = 10) randomized to 3-day periods of 2.5 h/day running/cycling or sitting and two days resting recovery, with crossover to the counterbalanced condition after a 3-week washout period. Fingerprick blood samples were collected pre- and post-exercise/sitting sessions during each 3-day period, and at 7:00 am the following two mornings (overnight fasted state). The Training Distress Scale (TDS) was administered at 7:00 am each of the five mornings in the lab.

Figure 2

Changes in the total Training Distress Scale (TDS) scores with exercise and rest conditions (interaction effect, p < 0.001). p-values, change pre-to-post change exercise compared to rest day.

Figure 3

Selected plasma proteins (from Table 2) increasing acutely each day of the 3-day exercise period compared to rest. (A) Lysozyme C; (B) Neutrophil elastase; (C) Neutrophil defensin 1; (D) Protein S100-A12; (E) Protein S100-A8; (F) Cathelicidin antimicrobial peptide; (G) Histone H2A types; (H) Histone H4. * p < 0.05, change pre-to-post change exercise compared to rest day.

Figure 3

Selected plasma proteins (from Table 2) increasing acutely each day of the 3-day exercise period compared to rest. (A) Lysozyme C; (B) Neutrophil elastase; (C) Neutrophil defensin 1; (D) Protein S100-A12; (E) Protein S100-A8; (F) Cathelicidin antimicrobial peptide; (G) Histone H2A types; (H) Histone H4. * p < 0.05, change pre-to-post change exercise compared to rest day.

Figure 3

Selected plasma proteins (from Table 2) increasing acutely each day of the 3-day exercise period compared to rest. (A) Lysozyme C; (B) Neutrophil elastase; (C) Neutrophil defensin 1; (D) Protein S100-A12; (E) Protein S100-A8; (F) Cathelicidin antimicrobial peptide; (G) Histone H2A types; (H) Histone H4. * p < 0.05, change pre-to-post change exercise compared to rest day.

Figure 4

STRING protein–protein interaction graph using immune-related proteins listed in Table 2. The thickness of the network lines indicates the strength of data support (https://string-db.org).

Figure 5

Selection process to determine the protein cluster (N = 13) associated with functional overreaching (FOR).

Figure 6

Selected plasma proteins increasing during day 1 and/or day 2 of recovery from the 3-day exercise period compared to rest, but not acutely immediately post-exercise. (A) Serum amyloid A-4 protein; (B) Myeloperoxidase; (C) Corticosteroid-binding globulin; (D) Complement C4B; (E) Complement component C8 gamma chain. * p < 0.05, change pre-to-post change exercise compared to rest day.

Figure 6

Selected plasma proteins increasing during day 1 and/or day 2 of recovery from the 3-day exercise period compared to rest, but not acutely immediately post-exercise. (A) Serum amyloid A-4 protein; (B) Myeloperoxidase; (C) Corticosteroid-binding globulin; (D) Complement C4B; (E) Complement component C8 gamma chain. * p < 0.05, change pre-to-post change exercise compared to rest day.

Figure 7

STRING protein–protein interaction graph using immune-related proteins listed in Table 3. The thickness of the network lines indicates the strength of data support (https://string-db.org). P01834 (Ig kappa chain C region) and P04220 (Ig mu heavy chain disease protein) for humans were not listed in STRING.