*Article* **Blood Biomarkers Variations across the Pre-Season and Interactions with Training Load: A Study in Professional Soccer Players**

**Filipe Manuel Clemente 1,2,\*, Francisco Tomás González-Fernández 3,4, Halil Ibrahim Ceylan 5, Rui Silva 1, Saeid Younesi 6, Yung-Sheng Chen 7, Georgian Badicu 8, Paweł Wola ´nski <sup>9</sup> and Eugenia Murawska-Ciałowicz <sup>10</sup>**


**Simple Summary:** Sports training may impact the variations of biomarkers in soccer players. Twentyfive professional soccer players were assessed twice in the season for their hematology and biochemical status, while training loads were monitored over the season. Relationships between changes in biomarkers and accumulated training loads were tested. Results revealed that that intense training in the pre-season period leads to decreases and increases in different hematological and biochemical markers.

**Abstract: Background:** Pre-season training in soccer can induce changes in biological markers in the circulation. However, relationships between chosen hematological and biochemical blood parameters and training load have not been measured. **Objective:** Analyze the blood measures changes and their relationships with training loads changes after pre-season training. **Methodology:** Twenty-five professional soccer players were assessed by training load measures (derived from rate of perceived exertion- known as RPE) during the pre-season period. Additionally, blood samples were collected for hematological and biochemical analyses. **Results:** For hematological parameters, significant increases were found for platelets (PLT) (dif: 6.42; *p* = 0.006; d = −0.36), while significant decreases were found for absolute neutrophils count (ANC) (dif: −3.98; *p* = 0.006; d = 0.11), and absolute monocytes count (AMC) (dif: −16.98; *p* = 0.001; d = 0.78) after the pre-season period. For biochemical parameters, there were significant increases in creatinine (dif: 5.15; *p* = 0.001; d = −0.46), alkaline phosphatase (ALP) (dif: 12.55; *p* = 0.001; d = −0.84), C-reactive protein (CRP) (dif: 15.15; *p* = 0.001; d = −0.67), cortisol (dif: 2.85; *p* = 0.001; d = −0.28), and testosterone (dif: 5.38; *p* = 0.001; d = −0.52), whereas there were significant decreases in calcium (dif: −1.31; *p* = 0.007; d =0.49) and calcium corrected (dif: −2.18; *p* = 0.015; d = 0.82) after the pre-season period. Moreover, the Hooper Index (dif: 13.22; *p* = 0.01; d = 0.78), and all derived RPE measures increased after pre-season period. Moderate-to-very large positive and negative correlations (*r* range: 0.50–0.73) were found between the training load and hematological measures percentage of changes. Moderate-to-large positive

**Citation:** Clemente, F.M.; González-Fernández, F.T.; Ceylan, H.I.; Silva, R.; Younesi, S.; Chen, Y.-S.; Badicu, G.; Wola ´nski, P.; Murawska-Ciałowicz, E. Blood Biomarkers Variations across the Pre-Season and Interactions with Training Load: A Study in Professional Soccer Players. *J. Clin. Med.* **2021**, *10*, 5576. https://doi.org/ 10.3390/jcm10235576

Academic Editor: David Rodríguez-Sanz

Received: 3 October 2021 Accepted: 24 November 2021 Published: 27 November 2021

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**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

and negative correlations (*r* range: 0.50–0.60) were found between training load and biochemical measures percentage of changes. **Conclusions:** The results indicated heavy physical loads during the pre-season, leading to a decrease in immune functions. Given the significant relationships between blood and training load measures, monitoring hematological and biochemical measures allow coaches to minimize injury risk, overreaching, and overtraining.

**Keywords:** soccer; performance; biology; workload

#### **1. Introduction**

Elite soccer has intermittent characteristics that require players to frequently engage in a high level of aerobic and anaerobic capacity [1]. Average VO2max values achieved by soccer athletes can reach up to approximately 63 mL/kg/min. While, maximal aerobic speed (MAS) can reach up to 17 km/h [2]. Professional soccer players have to perform low-intensity activities interspersed with high-intensity short explosive actions during training and matches [3].

Indeed, modern soccer is characterized by increasingly demanding physical activities during both training sessions and matches [4]. In fact, professional players can cover up to 7000 m of total distances (TD) in a single training session, and approximately 13,000 m during a match [5]. From the above-mentioned TD volume, players are required to cover significant distances in different high-intensity velocity thresholds, such as high-intensity running (HIR), high-speed running (HSR), sprints, and accelerations and decelerations [6,7]. Furthermore, different positions in the field require different physical demands. Therefore, it is essential to consider not only the biological individuality of each player, but also the physical demands of each position on the field [8].

As mentioned above, the pre-season is considered a critical period as, overall, players need to improve their fitness levels after the offseason period [9]. The detraining effects of the offseason period are accompanied by impairments in both physical and skills performance, that may be more pronounced if there is no individualized training program during the offseason [9,10]. Despite that, a study conducted on 23 elite soccer players showed improvements of approximately 8% in their aerobic and anerobic performance after a pre-season period [11]. Furthermore, physical and physiological changes during the in-season can be dependent on the physical and physiological status observed at the beginning of the season [12]. However, a recent study showed that improvements in aerobic fitness after a pre-season period may not happen in a linear fashion as the authors found that fitness changes after the pre-season have a great variability between different seasons [13].

For such reasons, it is of paramount importance to monitor internal load measures on a daily basis. There are several psychometric measures, including fatigue, stress, soreness, quality of sleep factors, and their respective Hooper Index score (sum of the four factors), to monitor the well-being status of each player on a daily basis [14,15]. The Hooper Index score has been associated with the training load in soccer, showing its usefulness for practice [16]. In fact, a recent study conducted on nine professional soccer players revealed that the Hooper Index score had lower typical errors than the heart rate variability [17]. Thus, its usefulness seems to be promising in monitoring player's fatigue during a soccer season. Furthermore, the load monitoring can be daily applied using subjective measures. Those measure are based on the rate of perceived exertion (RPE) scales to obtain an indicator of global internal load of soccer training sessions, such as the session-rate of perceived exertion (s-RPE) [18]. In addition, other authors have started to use other RPE measures in their investigations, such as the sRPE general, sRPE breath, and sRPE neuromuscular [19,20]. These new s-RPE measures can determine the subjective perception of exertion on different body structures [20]. However, Los Arcos et al. [21], revealed no relationships between sRPE general, sRPE breath, and sRPE neuromuscular with changes in aerobic fitness.

Besides the common influencers of aerobic fitness (e.g., ventilatory kinetics, cardiac process, neuromuscular status), other hematological and biochemical parameters assume a preponderant role in athletes' performance [22]. However, there is incongruent evidence regarding the effects of acute and/or chronic training stimulus on hematological parameters, such as hemoglobin (Hb), red blood cells (RBC), and hematocrit (Ht) [23]. It seems that there is a trend to observe increases in the above-mentioned hematological parameters after a period of soccer training, especially during the preparation phase [24]. The Hb, RBC, and Ht are important hematological parameters since they are linked to the player's aerobic capacity, which is one of the physical aspects most trained during the pre-season [23,25]. In the case of biochemical parameters, they represent an important role for the monitoring of an athlete's responses to the training loads imposed [26]. For instance, cortisol and testosterone levels represent good markers of training stress, with cortisol being associated to catabolic processes and testosterone to anabolic processes [27]. In fact, a study conducted on 25 soccer players affirmed that the high training volumes during the pre-season period causes a decrease in testosterone levels and an increase in cortisol levels [28]. Thus, in consequence of high training loads imposed, the athletes enter in a catabolic state that impairs physical performance [29].

These facts reinforce the need to be aware of other possible biochemical associations with the imposed training loads on athletes, especially during the pre-season period, where higher loads are imposed to athletes. Moreover, considering the injury rate during a soccer season, the neutrophils, monocytes, and eosinophils have an important role in the reaction to inflammation, acting as a defense through the process of phagocytosis. Lymphocytes and basophils also constitute a major importance in the immune system and in the defense against acute viral and bacterial infections [30], given that their relationships with training loads can be useful in relation to primary prevention of injuries. To the best of our knowledge, there is no study addressing different blood biomarkers variations and their interactions with different external load measures during the pre-season period. For those reasons, the purpose of this study is twofold: (i) Analyze the variations of chosen biological markers before and after the pre-season period and (ii) analyze the relationships between variations of biological markers and workload imposed on the players.
