**4. Discussion**

The purpose of this study was twofold: (i) Analyze the variations of 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. To the best of our knowledge, there is no study that addresses different blood biomarkers variations and their interactions with different internal load measures during the pre-season period. The major findings of the present study indicate that the Hooper Index, RPE (general, breath, and neuromuscular), and sRPE (general, breath, and neuromuscular) increased progressively after the pre-season. Likewise, PLT, creatinine, CRP, ALP, cortisol, and testosterone increased, whereas ANC, AMC, calcium, and calcium corrected decreased significantly after the pre-season period. Furthermore, several significant relationships were found between blood biomarkers, training load parameters (RPE and sRPE), and psychometric variables (the Hooper Index, fatigue, stress, soreness, and quality of sleep).

The pre-season is widely accepted to be the period with a high training load [38,39], and concomitant augmented risk of sustaining injuries [40]. High-quality pre-season soccer training plays a role not only in improving physical fitness (aerobic capacity), but also in injury prevention [41]. The monitoring of blood biomarkers before and after pre-season plays a role in increasing positive adaptation, and reducing the risk of injuries, illness, and overreaching caused by stress factors that occur during soccer matches over a season [42]. In our study, significant increases were found in training load parameters, the Hooper Index, RPE (general), RPE (breath), RPE (neuromuscular), sRPE (general), sRPE (breath), and sRPE (neuromuscular) after pre-season compared to before pre-season. Recent studies have frequently shown that internal or external workload indices [43]. In addition, the Hooper Index parameters were found to be higher during the pre-season period compared to other periods of the season [44,45]. The increase in training load parameters in the pre-season is usually due to the progressive overload principle of training, to prepare the players to meet the physical demands of the upcoming season [39].

Furthermore, the present study revealed a significant increase in PLT after the preseason period. In the literature, there are studies with different results regarding the decrease [22], increase [46–48], or lack of changes [42] in PLT after long-term intensive soccer or different kinds of exercises. Michail et al. [46] revealed a similar conclusion to the results of the present study, as they found a significant increase from 231 × 103/μL to 244 × 103/μL of the PLT amount after the soccer intensive exercise intervention program. Moreover, a study conducted on 13 male soccer players, with significant augment in PLT levels (209.76 ± 33.83 to 249.76 ± 61.09 × 103/μL) was noted following 2 weeks of pre-tournament moderate-to-high intensity training period [49]. Contrary to our study, Ozen et al. [50] found an increase in PLT after the pre-season training period in welltrained young soccer players. However, their reported increase (pre: 205.57 ± 54.94, post: 214.85 ± 23.12) was not significant.

The reason for a high number of circulating PLT in the blood (thrombocytosis) after intense soccer exercise can be explained by epinephrine hormone secration, which has the ability to cause a strong contraction of the spleen (the storage area of one-third of the body's PLT), and may play a role in the increase in PLT after exercise [51]. Likewise, it was declared that the mechanisms related to the increase in PLT after high intensity exercises were not clear [48]. However, those increases might be due to increased PLT production by cells in the bone marrow, and decreased removal of PLT from the blood, which was one of the functions of the spleen [48]. Another possible mechanism is shear and oxidative stress, which can activate PLT. Exercise-activated PLT contribute in growth factors liberation and proinflammatory mediators [52]. As in this study, an increase in PLT after intense exercise may also be associated with an improvement in performance. It was previously reported that hyperactive PLT have some pleiotropic effects on endurance sport performance, both by releasing ergogenic mediators and triggering an increase in performance-enhancing substances, such as nitric oxide into the circulation [53].

Regarding hematological parameters, our study revealed that the ANC and AMC significantly decreased after the pre-season period. Consistent with our findings, Heisterberg et al. [54] indicated that the numbers of circulating monocytes decreased at the end of a training season. In other study, it was noted that there was an increase in neutrophils and a decrease in lymphocytes after short periods of pre-tournament training [49]. Ozen et al. [50] reported no significant differences in subpopulations of leukocytes (lymphocytes, neutrophil, monocyte, and basophil percentage) after the pre-season period in young male football players. In a previous study, which was not consistent with the findings of our study in terms of neutrophil, an increase in neutrophil counts was found after regular and vigorous soccer exercises, and it was suggested that this situation was associated with minor inflammatory events [22]. In addition, contrary to our study, Dias et al. [55] notified an increase in total leukocyte, neutrophil, and monocyte counts, whereas lymphocytes

reduced by the end of the season in volleyball athletes, and they also claimed that the increase in total neutrophils and monocytes might be due to muscle tissue remodelation, resulting from potential damage induced by training load and competition.

Furthermore, the present study revealed that decreases in ANC and AMC after the pre-season period may be related to the timing of blood collection after the last exercise session. In our study, there was a time of 12 h between the last training session, and the second blood draw (after the pre-season). This may have caused a short-term temporary suppression of the immune system in soccer players after the last training session, i.e., the previous day's acute high-intensity exercise. This situation is defined as "open window" immunological phenomenon in the literature [56–58]. Moreover, previous studies showed that high-intensity exercises could lead to a short-term, acute inflammatory response [59–62]. Another study also supports the findings of the present study, in which the authors alleged that intense endurance activities decreased neutrophils, and monocytes in athletes, and this condition was related to the depression of the immune systems, which triggered an increased the risk of disease or infection, especially the pre-season period [63]. Lastly, regarding the leukocyte count and subpopulations in the pre-season period, the present study shows that there is no pathological condition, only the decreases in ANC and AMC may be associated with timing of blood collections. It can also be suggested that training in pre-season do not produce chronic effects on immune function and susceptibility to infection.

Creatinine is a metabolic product of a creatine breakdown during energy metabolism. The serum creatinine level is a known parameter for evaluating renal function in clinical medicine, and is used as an indicator of general health status and water-electrolyte balance in sports medicine [64]. The present study revealed that there was a significant increase in creatinine after the pre-season period. Our results are not consistent with some studies. For instance, Meyer & Meister [65] found only minor changes in creatinine levels in professional football players over a season. Another study revealed that there was no significant change in the serum creatinine level of rugby players before and after the training camp [66]. Furthermore, Andelkovi´c et al. [22] affirmed that serum creatinine levels in soccer players decreased significantly throughout the study, which might be related to the increase in training and competition workloads during the half competitive season. Prior studies on soccer player demonstrated that creatinine levels were higher in players with greater training and match loads (cumulative match-time) throughout the season [64], and also increased post-match in comparison with pre-match values due to the high intensity of the performance during the match [67]. Regarding creatinine as a by-product of muscle contraction, its rise after a match or higher training load, especially the pre-season period, could be due to the deterioration of muscle tissue [67]. Additionally, another study asserted that the increase in plasma creatinine after intense soccer exercise stemmed from the creatinine release from working muscles, dehydration, and/or reduction in renal blood flow and glomerular filtration rate [68]. In our study, there were negative large correlations between the percentage of change of urine color and percentage of change of creatinine. After creatinine is used by the muscles, it is filtered by the kidneys, and excreted in the urine, based on this information, this study reveals that urine is the determinant of the percentage change in creatinine.

Increased levels of oxidative stress are closely associated with markers of muscle damage with high inflammation [59]. CRP is the most common inflammation molecule of the body's acute phase response, and it increases the inflammatory response to various stimuli that initiate the acute phase response [47,69,70]. In our study, significant increases were found in CRP values after post-pre-season compared to pre-pre-season. The CRP level has been found to increase during the inflammed state, that is, after intense exercise [71,72]. Significant increases in CRP after a soccer match in amateur soccer athletes was previously shown [47]. Mohr et al. [73] also found CRP values before (0.9 ± 0.1 mg/L), and after (1.3 ± 0.0 mg/L) the preparation period in professional soccer players. The studies mentioned above support the results of our study. However, these results differ from the

study published by Radzimi ´nski et al. [70], where it was found that elevated CRP values were not detected in soccer players during a pre-season sports camp (pre: 1.44 ± 0.7 mg/L, post: 0.83 ± 0.34 mg/L), i.e., above the reference range (<5.0 mg/L).

It was recently determined that decreases in CRP levels of futsal players, also asserted that reductions in the CRP level indicated that players adapt to the training load applied throughout the competitive season [59]. Radzimi ´nski et al. [70], emphasized that inflammation in the bodies of pre-season soccer players might be the result of misuse of high-intensity training loads in a short time. On the other hand, a previous study stated that GPS variables associated with high-intensity activities, such as running speeds, accelerations, and decelerations were useful markers for detecting muscle damage or inflammation [74]. Similarly, Coppalle et al. [69] found a significant and very large correlation between total distance covered (>20 km/h) and CRP after the pre-season period in professional soccer players. The increment in CRP after the pre-season may be related to the frequent use of high-intensity activities in training during this period. However, the present study exhibited that this increase in CRP does not seem to reflect a pathological condition. Finally, it was pointed out that the rise in CRP after intensive exercise could be the result of mechanisms, such as the inflammatory response to injuries or agents (interleukin-6, i.e., the main stimulator of CRP secretion) that might be associated with elevated inflammation in athletes [47].

Moreover, the present study showed that alkaline phosphatase (ALP) significantly increased after the pre-season period. In the literature, some studies showed that ALP increased after intense soccer exercise [68,75], while some studies showed that no significant change in the ALP level of players with a higher training load over a season [64]. As in our study, the increase in ALP after intense soccer exercises might be associated with the result of some leakage from skeletal muscles of enzymes that play a role in the sustained release of ATP, and catabolize amino acids during exercises [68]. In addition, the increase in ALP after the pre-season period in our study may be explained by another study [76], as the authors suggested that the elevation in ALP levels reflected liver increased activity for gluconeogenesis, lipid peroxidation, and increased bone turnover triggered by the duration and intensity of exercise. Considering the CRP and ALP parameters related to inflammation, the physiological increase in CRP and ALP may be the result of acute high-intensity exercise [61] performed the day before blood collections. However, the present study demonstrated that pre-season intense soccer training does not cause any chronic effect on susceptibility to inflammation.

Calcium is a necessary mineral for proper growth, maintenance, and repair of bone tissue, nerve conduction, blood coagulation, and regulation of muscle contraction. Serum calcium level is tightly arranged by calcitonin and parathyroid hormone, independent of acute calcium intake [77–79]. In our study, statistically significant reductions were found in the calcium and calcium corrected after the pre-season period. The study of Mashiko et al. [66] does not coincide with our results, as they reported that there was no significant difference in the serum calcium level of rugby players after 20 days of preseason intensive training. In our study, the decrease in calcium after an intense pre-season period can be explained as follows; calcium may leak into the tissue to create muscle contractions during exercise, so blood levels may decrease after intense exercises. In the report published by the UEFA expert group on nutrition in elite soccer, a daily calcium intake of 1300–1500 mg/dL is recommended for professional soccer players to optimize bone health in cases of relative energy deficiency in sports [77]. Accordingly, a recent study determined that soccer players did not meet their daily calcium needs in the preseason period [79]. Given the importance of calcium for bone health, reductions in calcium concentration may result in decreases in bone mineral density, which can elevate the risk of injury to players throughout the season.

Cortisol and testosterone hormones play a role in catabolic and anabolic processes [80], are frequently used in studies as training stress markers, and these markers are closely associated with overreaching and overtraining syndromes [28,81]. The results found in the present study demonstrated that both cortisol and testosterone were significantly augmented in response to a soccer pre-season period. Di Luigi et al. [82] reported that salivary cortisol and testosterone level increased after an acute response to soccer exercise in young soccer player. Similarly, Muscella et al. [83] observed increases in both testosterone and cortisol levels after an intense training period in soccer referees. Nogueira et al. [84] remarked that testosterone increased, while the cortisol hormone decreased in futsal players after 4-weeks of pre-season. The same authors [84], noted that these results promoted an anabolic environment, which is also consistent with the finding of the study conducted by Perroni et al. [81]. Nevertheless, there are studies showing the formation of a catabolic environment (increases in cortisol, and decreases in testosterone levels) due to a high training load in the pre-season period [28,29,39,42]. It was reported that such a catabolic physiological environment could adversely affect various physical performance-related parameters such as speed, vertical jump height, and muscle strength throughout the season [28]. The T/C ratio is used to evaluate the balance between anabolic and catabolic activity [85,86], and represents a benefical tool in the early detection of overtraining [87]. The present study revealed that there were no significant changes in the T/C ratio after the pre-season training period. This result was supported by a previous study that showed that no significant changes in the T/C ratio after intense pre-season traninig in soccer players [88], and non-athletic men [83]. Contrary to our findings, recent studies observed significant reductions in the T/C ratio in response to a high volume of training sessions [39,89], and a period of congested match play [86,89] in professional soccer players. Similarly, another study demonstrated that a decrease equal or higher than 30% in the T/C ratio reflected state of catabolism, which resulted in a prolonged recovery time, fatigue, and deterioration of competitive soccer performance [90]. Additionally, our result was not similar to previous studies that reported that the T/C ratio increased significantly in team sports athletes after the pre-season period [81,84,85,89]. As in the present study, Botelho et al. [88] stated that a significantly unchanged T/C ratio after the pre-season period was associated with a favorable response to the training load, and adequate coping with training stresses. The current study revealed that the T/C ratio, which did not change significantly, and the conversely significant increases in cortisol and testosterone, after the pre-season could be explained by an environment that reflects a dynamic hemostatic balance between an anabolic and catabolic process in muscle [81,83]. This is very important in terms of both the prevention of the risk of injury of the players, and the quality of their physical performance during the training and competition season. Nonetheless, considering the testosterone, cortisol, and T/C ratio, the training load distribution and the load-rest relationship are well adjusted during the pre-season period, and the players have responded adequately to the training load without the accumulation of fatigue. Additionally, they probably have not experienced overreaching and overtraining. Moreover, the current study showed that the percentage of change of sRPE breath was a predictor variable of percentage of change of testosterone. Consistent with the present study, Peñailillo et al. [91] reported that the rate of perceived exertion was positively related to the change in testosterone levels. Accordingly, another study found that a higher internal training load (RPE-based) triggered anabolic stimulus (that is increases testosterone secretion) which positively affected performance in professional soccer players [92].

The present study indicated that negative large correlations were observed between a percentage of change of sleep quality and percentage of change of albumin, and also a percentage of sleep quality was a predictor variable of percentage of change of albumin. Sleep needs and rest are important for rapid recovery, and preventing the risk of illness, injury, and bad-overreaching in the pre-season period [93]. The deterioration in sleep quality due to a higher training load can be observed in the pre-season period, which may negatively affect biochemical parameters, especially albumin [30,42]. A previous study showed that that high-volume running exercises, which were frequently performed during pre-season training, caused a high sweating rate, which led to blood thickening, and as a result, it triggered an increase in the amount of albumin in the blood. Furthermore, in our study, blood measurements were performed in the morning hours (08.00–10.00 a.m.). Moreover, it was indicated that the augment in serum albumin levels in morning hours was closely related to the fact that normal blood thickening was not restored by overnight rest after exercise [66].

In the literature, there are limited studies examining the relationship between internal load indices (RPE, sRPE), wellness parameters (hooper index), and blood biomarkers. This is the first study to examine the relationship between pre-season training load (RPE, sRPE, and the Hooper Index) and blood biomarkers. Still, regarding the relationships between blood biomarkers, training load parameters (RPE and sRPE), and psychometric variables found in the present study, it is suggested that the internal load and Hooper Index parameters are associated with markers of inflammation and muscle damage. Interestingly, Dias et al. [55] reported that immune variables, such as total leukocytes, neutrophils, and lymphocytes might be modulated by training loads and by tactical and physical components. Indeed, Coppalle et al. [69] indicated that muscle damage or inflammation indicators, such as lactate dehydrogenase were correlated to RPE values, and suggested that the fatigue-related muscle damage enzyme increased at high perceived exertion levels. The same findings were also observed in our study. However, further research is needed to generalize the results from this study.

The present study contains some limitations that should be emphasized. First, the number of participants in our study was not very large. Considering the relationship between nutrition and hematological/biochemical parameters, no determination was made regarding the nutritional status of players in the pre-season period. In other words, the food consumption of players was not followed nor were there even supplements taken in the period. Furthermore, no measurements were made regarding the injury rate of the players. The relationship between pre-season training load parameters and injury rate could be examined. Despite the limitations mentioned above, the present study is to first examine different blood biomarkers variations and their interactions with different internal load measures during the pre-season period. In the future, by elevating the number of participants, it is recommended to increase the number of studies to compare blood biomarkers taking into account the gender and age factor in teams in different leagues according to player positions during the pre-season or the entire season, and to examine the relationships between these biomarkers, training load, and injury rate.

#### **5. Conclusions**

The present study revealed that intense training in the preseason period leads to decreases (ANC, AMC, calcium, and calcium corrected), and increases (PLT, creatinine, CRP, ALP, cortisol, and testosterone) in different hematological and biochemical markers. The present study also showed several significant relationships between blood biomarkers, training load parameters, and wellness variables. Given that, training load distribution is of critical importance in the optimization of blood biomarkers, especially during the pre-season period. In addition, ensuring a balance between the training load and blood biomarkers in the pre-season period contributes to the maintenance of high level physical performance of players during the entire season, and to prevent the risk of injury, badoverreaching, and overtraining. Moreover, comprehensive monitoring of blood biomarkers in terms of hematological, nutritional, biochemical, muscle damage, and hormonal markers along with internal load indices and wellness measures can provide clearer insights into the mechanisms underlying players' performance throughout the season.

**Author Contributions:** Conceptualization, F.M.C.; methodology, F.M.C. and F.T.G.-F.; data collection: S.Y.; formal analysis, F.T.G.-F. and R.S.; writing—original draft preparation, F.M.C., F.T.G.-F., H.I.C., R.S., S.Y., Y.-S.C., G.B., P.W. and E.M.-C.; writing—review and editing, F.M.C., F.T.G.-F., H.I.C., R.S., S.Y., Y.-S.C., G.B., P.W. and E.M.-C.; supervision, F.M.C. All authors have read and agreed to the published version of the manuscript.

**Funding:** This work is funded by Fundação para a Ciência e Tecnologia/Ministério da Ciência, Tecnologia e Ensino Superior through national funds and when applicable, co-funded EU funds under project UIDB/EEA/50008/2020.

**Institutional Review Board Statement:** The study protocol was approved by the Scientific Committee of School of Sport and Leisure (Melgaço, Portugal) with code number CTC-ESDL-CE00118. The study followed the ethical standards of the Declaration of Helsinki.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study.

**Acknowledgments:** This study made is a part of one curricular unit of Master in Sports Training at Escola Superior de Desporto e Lazer, Instituto Politécnico de Viana do Castelo, Portugal.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

