Statistical difference from T0. \* Statistical difference from T1. § Statistical difference from T2. \$ Statistical difference from T3. <sup>A</sup> Statistical difference from 2018/2019 season.

#### *3.5. Physical Performances*

No significant changes were observed for the CMJ Test (Table 7). However, the between-period comparison revealed significant differences because the changes associated with the COVID-19 lockdown were significantly worse than those occurring during the 2018/2019 competitive season (Table 7).

**Table 7.** Differences (∆) in blood lactate concentrations and CMJ between each timepoint in football players all for 2018/2019 and 2019/2020. *p*-values < 0.05 obtained by *t*-test show statistical differences in ∆ values.


Analysis of blood lactate concentrations during the competitive seasons showed a significant decline, but a significant increase was observed following COVID-19 lockdown. Consequently, between-period differences were significant when the COVID-19 lockdown period was compared with the 2018–2019 competitive period (Table 7).

#### **4. Discussion**

Due to physiological and performance adaptations to training, professional football players are subjected to several alterations in health [16,17] and performance [18] throughout the course of the season. Although a variety of research has investigated performance testing and/or observational approaches to explore the relationship between training load and training outcomes (e.g., acute responses, chronic responses, and injuries) [19], limited information is available regarding blood parameters (e.g., iron storage and hormonal environment) of elite football players from the same team, before and after a different period of match play and training, even more since March 2020, when the COVID-19 pandemic forced most activities in Italy, including football, to stop. During lockdown, players could only train at home, with limited evidence regarding the effect of this period [20]. Therefore, this study aimed to investigate the effect of COVID-19 lockdown on professional football players. Thus, we describe the seasonal changes in anthropometric and body composition indicators, hormonal status, and performance in a professional football team, during two different sporting seasons: the regular 2018/2019 season and the 2019/2020 season that was stopped from March to May and finished on 2 August 2020.

Data from anthropometric, blood values, and hormonal parameters showed differences between the two seasons, proving that the forced stop period affected the physical and physiological state of professional football players. According to numerous studies, anthropometric and body composition indicators are important factors, which can predict the specific footballer's performance, already in his adolescence [21]. In the 2018/2019 season, without any interruption, there was a constant decrease in weight, BMI, and percentage of fat mass and an increase in fat-free mass. In the 2019/2020 season that underwent the forced stop, however, the same trend was recorded but up to T1. In T2 and T3 periods, there was an increase in weight and BMI, while the percentages of fat mass and lean mass remained almost constant.

It is well established that training may alter homeostasis, hematological parameters included. Hb is a key determinate of oxygen transport and consumption [22], which is related primarily to aerobic capacity [23]. Elevated Hb is generally associated with an increase in blood oxygen transport capacity, while an increase in Ht increases blood viscosity [24]. Thus, it seems beneficial to monitor football players' Hb and Ht parameters. Several studies showed decreases in Hb and HT values after periods of intense training or competition [25]. These declines are known as an adaptation to training [26]: erythrocytosis during exercise induces an increase in the absolute concentration of Hb [16], but this mechanism is masked by a rise in plasma volume (PV) [27]. PV expansion compensates for the negative effects of acute blood concentration induced by intense training. In fact, an increase in aldosterone levels and osmotically active plasma proteins, as well as a decrease in the activity of urodilatin, eventually lead to fluid retention and PV increment [28]. This increase in PV is a first sign of overtraining [27].

To date, only a few studies have evaluated Hb and HT values during an entire competitive season in football players [9,29]. Particularly, Silva et al. [30] observed that the Hb and HT of Brazilian football players increased significantly after 12 weeks of training. These authors postulated that such alterations were due to the plasma volume decrement observed after the football-training program. A study of Saidi et al. [9] suggested a significant change in various hematological parameters with negative effects on physical fitness during 6 weeks of congested match play. In contrast, Heisterberg et al. [31] and Rago et al. [29] recorded no significant changes in Hb and HT levels over a 6 month period in which the training and match load varied considerably. Ostojic et al. [32] found a significantly higher HT at preseason compared with other sampling periods, and no other differences were found between any of the hematologic variables during the whole season. These differences in the results obtained in these studies could be due to psychological factors, players' diet [33], and/or differences between players' effective match time.

In the present study, we hypothesized that in the period of congested games (between T3 and T4), in which the championship resumed after the COVID-19 lockdown, the football matches would have negatively affected the plasma volume and hematological parameters. Instead, apart from a slightly decreased Hb during the COVID-19 season, we did not find significant changes in the hematological variables tested. In addition, it should be remarked that during the COVID-19 season (i.e., when the training load is discontinuous), the hemodilution was absent. This phenomenon observed during the 2018/2019 season could be a favorable adaptation to training because decreased blood viscosity allows greater cardiac output. Instead, erythrocytes, Hb, HT, and plasma volume values decreased during the 2018/2019 season, without any interruption. Regarding erythrocytes, in agreement with our results in the 2018/2019 season, many studies have suggested that the number of erythrocytes decreased at the end of a competitive period [9]. In general, erythrocytes, Hb, and HT decrease after endurance training [34]. This is mainly caused by PV expansion [35]. In fact, Silva et al. [30] were able to show that the altered percentage of erythrocytes significantly correlated with plasma volume change (i.e., reductions) during the 12 week football training program. Among others, adequate serum iron levels seem to be the main factor for optimal hemoglobin production, maximal oxygen uptake (VO2max), and high sports performance in football [36]. Several investigators suggested that the iron status of elite athletes also varies during the season because of different training regimes [37]. The iron depletion in top-level football players based on low serum ferritin levels could be crucial for predicting optimal physical performance [38]. In fact, it seems that ferritin values decrease with the training load, suggesting that ferritin could be a marker of training tolerance in endurance athletes [39]. In accordance with other studies [39], in our study in footballers, ferritin showed a constantly decreasing trend from the initial phase of both seasons. Thus, the serum ferritin level strongly decreased, especially during the first part of the regular season (T1–T2). Iron is essential for normal cell biology. However, excess iron might be potentially harmful, as it can catalyze the formation of toxic reactive

oxygen species. Therefore, the decreased levels of serum iron, observed along both seasons considered, might be an adaptive response [40].

Vitamin D, mainly synthesized by the skin when exposed to ultraviolet B radiation (UVB), is involved in several physiological processes as the maintenance of calcium, phosphate, and iron homeostasis [41]. During winter, vitamin D deficiency can occur in up to 50–80% of the population [42]. Although an optimal vitamin D level helps to maintain the musculoskeletal system efficiency [43], studies on athletes highlighted a surprisingly high prevalence of vitamin D insufficiency, both in outdoor and indoor disciplines [44]. Thus, even outdoor training (such as by football players) is not protective against vitamin D deficiency [45] and many studies performed in European football players during the winter season showed serum vitamin D levels below the normal range (defined by the latest guidelines as 30–50 ng/mL) [46,47]. Our data confirmed the high prevalence of low serum concentrations of vitamin D in professional male athletes [48] in winter. In both seasons, the mean concentration of Vitamin D was insufficient (defined as a serum level of 20–30 ng/mL) in T3 (in March) and in the COVID-19 season also in T2. Surprisingly, this was also true in the 2018/2019 season, when football players trained outdoors 2 h a day.

Creatine kinase (CK), cortisol, testosterone, and the testosterone/cortisol ratio (T/C) have been used to assess athletes' response to training load [49,50]. Precisely, CK levels have been used to monitor muscle damage and post-match neuromuscular fatigue in elite football players [51] and other sports [49,50]. Most studies have reported data from singlematch experiments [52] or short-term studies [53] and much less information is available on long-term studies during the entire season in elite professional football [54]. In the present study, in T2, T3, and T4 of both seasons, CK values were slightly above 270 U·L −1 , set as the highest reference value for the general population. High values of CK have been suggested as a symptom of overreaching or overtraining [55] and unusually higher values of CK have been routinely measured in blood samples from football players (until 1492 U/L) [55]. This may be related to the nature of football training and playing involving a great deal of weight-bearing activities, which include eccentric (lengthening) contractions of the leg muscles [55]. In addition, football playing can induce muscle damage due to mechanical impact with other players. Finally, football training and competition are often performed under severe environmental conditions, and football games are among the longest (90 min) and most energy-demanding sporting activities [56].

Plasma cortisol and testosterone are sensitive to training periods that differ in volume and intensity and to the frequency of matches during the competitive period [10]. Many factors influence this delicate hormonal balance, not only training workloads, training schedules, and competition factors but also psychological stress. If the physical demands of training and competition are too great, one might assume that catabolic activities will predominate. However, when the body successfully copes with the demands, the anabolic metabolism can improve the performance during different periods of the competitive season [57]. Thus, testosterone and cortisol values can be considered important parameters that help to evaluate the influence of these factors because of the balance between anabolic and catabolic processes [58]. Some studies have identified a significant increase in cortisol concentration, in football players [10,59], due to an increased training intensity. This is probably caused by hyperresponsiveness of the hypothalamic–pituitary–adrenal (HPA) axis due to a physiological adaptation of the neuroendocrine system to training [60]. In addition, a substantial increase in cortisol, at the end of the football season, may be explained by tiredness due to the higher number of matches placing a higher physical load, as well as psychological pressure, on the players [61]. The results obtained from this study show that the hormonal concentrations of cortisol and testosterone are higher during the 2018/2019 season, in which the football players trained regularly and played all the official matches. The increase in plasma cortisol during the 2018/2019 season may represent a typical homeostatic adaption process to the soccer training and to a stressful environment and competitions [10], with an increment by 20% during the T0–T2 timepoint. In contrast, other studies have shown a significant decrease in cortisol concentration

after long periods of training in elite football players [16,62]. In accordance with both Saidi et al. [9] and Requena et al. [18], we observed no changes in cortisol concentration in elite football players, during the 2019/2020 season.

Though we would have expected a significant increase in plasma cortisol after the lockdown, it should be noted that the absence of a significant change in its concentration could be attributed to the lack of stress due to the weekly official sports competitions, rather than the high level of fitness of the players and/or their usual high daily and weekly training volume over the course of the season. Several studies have documented the effects of COVID-19 on the psychological stress experienced in the general population [63], and it is known that uncontrolled stressors activate the HPA axis: through the association of the cortex, amygdala, hippocampus, and adrenal glands, blood cortisol increases [64]. As high levels of stress can have a detrimental impact on everyday life and mental and physical health, there would be a need to examine and diagnose psychological problems and deteriorating mental health among professional athletes during the COVID-19 pandemic. In fact, training restrictions and competition avoidance, due to COVID-19 lockdown, decreased competitions performance, substantiating the contribution of cognitive distress to the overall perception of effort and to performance outcome [20]. However, previous studies have reported that COVID-19 preventive measures did not provoke changes in the levels of anxiety, stress, and symptoms of depression in professional footballers [65,66]. Our study confirmed that the professional players cope very well with the changes due to COVID-19, with no noticeable changes in cortisol, indicating good psychological adaptation. Therefore, from a practical point of view, training regimens and healthy behaviors during pandemic crises could be introduced as standard habits for health and well-being. In the pandemic period in response to social distancing, coaches and athletes had to adopt a constructive problem-solving attitude and make structural changes to the training environment. Although the time spent for sport-specific training was reduced, individualized home-based training was implemented; this could have turned into improved training conditions. Indeed, at the Tokyo 2020 Olympics, swimmers' performance trend was maintained despite the unprecedented characteristics of the previous period of preparation, demonstrating that the supposed effects of the COVID-19 lockdown on elite athletes' performances were not apparent. Financial, social, psychological, scientifical, and technological support environments of Olympics participants could safeguard the subsistence of performances even in the case of periods of difficulty never faced before [67].

The change in anabolic hormonal concentrations could be due to interactive modifications of various endocrine parameters, and to the effects of modifications of the hypothalamic–pituitary axis on the testicles and adrenal glands. The training-induced increases in serum testosterone reflect the anabolic activity increment related to the volume of strength training and to physical performance improvements [68]. In our study, we observed that after an intense training period, testosterone concentrations increased in both seasons, remaining in normal ranges, as also reported by others [10,69]. However, decreases in testosterone have also been reported after long-term football training, perhaps due to excessive training intensity, accumulated fatigue, or physical fitness declines [9]. The testosterone concentration decrement found in T3 during the COVID-19 season might be due to tiredness caused by the high number of matches played and to the psychological pressure at the end of the lockdown period. Regarding the T/C ratio, results showed an increase from T0 to T1, during the 2018 season. According to some studies, the T/C value increment is indicative of a good training setting and can reflect physical performance improvements [70,71]. However, in contrast to these investigations, other recent studies showed a significant decrease in the T/C ratio in professional football players [9,61], as being due to neuromuscular fatigue caused by an increase in training intensity that might be related to physical performance declines [9]. The divergence of these results can be explained by differences in training programs (frequency, duration, and intensity) and/or participants' expertise level (training history). These two hormones appear sensitive to the intensity and volume of football training and other factors such as fatigue or mood and, if

properly interpreted, could provide a tool for monitoring workload and fitness. Despite the least amount of work carried out during the COVID-19 period, aerobic fitness, as measured by the Mognoni test, improved after the lockdown. There was also a significant difference between the same timepoints of COVID-19 and 2018–2019 seasons.

In addition to training volume, training intensity is another key factor that may influence aerobic adaptations [24]. Specifically, high-intensity interval training can elicit significant improvements in highly trained athletes, whereas additional submaximal endurance exercise does not seem to lead to further changes [23]. Additionally, in normal circumstances (i.e., during the competitive period), the training volume is not entirely dedicated to the improvement of players' physical capacities, because technical-tactical drills are performed more frequently. This kind of exercise may not always reach an adequate intensity to elicit positive adaptations, whereas during the COVID-19 lockdown period, training activities were mainly focused on physical conditioning, which, in turn, probably induced superior positive aerobic adaptations.

The findings from this study are related to only one Italian football team, but different restrictions in other countries might have had different effects on football players' physical performance and psychological implications.

In addition, different home-based training strategies (including the different equipment) could result in different adaptations, in biological and physical performance, on football players.

#### **5. Conclusions**

This study was conducted to provide reference data in professional football players, especially under stressful conditions, such as the pandemic period. Despite the spontaneous variability in most parameters, there were significant changes during both seasons in hematocrit, hemoglobin, iron, ferritin, vitamin D, and testosterone.

However, from a medical perspective, none of the changes were considered clinically relevant to a player's health or training status, because all were within normal values and most likely typical modulations of homeostatic concentrations in response to a stressful environment, training, and competition. In addition, this study showed that the training volume during home confinement in the COVID-19 period was probably insufficient to allow professional football players to maintain the jumping power of competitive periods. These changes might be not relevant enough to possibly interfere with clinical decisions in football players.

**Author Contributions:** Conceptualization, A.M., A.B., C.M.d.S. and S.M.; methodology, G.M. and D.Z.; software, G.M.; validation, A.M., A.B., C.M.d.S. and S.M.; formal analysis, A.M. and S.M.; investigation, G.M. and D.Z.; data curation, A.M. and D.Z.; writing—original draft preparation, A.M.; writing—review and editing, S.M. and C.M.d.S.; visualization, A.M., A.B., C.M.d.S. and S.M.; supervision A.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was approved by the Institutional Review Board (I.R.B.) of the Department of Biological and Environmental Science and Technologies (Di.S.Te.B.A., (protocol code N-1/2021 and 7 January 2021), and all experiments were conducted in accordance with the 2013 Helsinki declaration and its later amendments. All data were anonymous and confidential in line with new European data protection laws (Regulation GDP. Regulation EU 2016/679 of the European Parliament and of the Council of 27 April 2016. Off J Eur. Union. Available online: https://eur-lex.europa.eu/legal-content/EN/TXT/PDF/?uri=CELEX:32016R0679, accessed on 10 January 2021).

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

**Data Availability Statement:** Data available on request due to restrictions, e.g., privacy or ethical.

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

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