The experiment lasted for 6 weeks and covered the preparation period for the summer season (Figure 1). The athletes trained on an everyday basis (approximately 2 h/d) with an official soccer game on Saturday/Sunday. Additionally, twice a week, the players performed a strength and conditioning training session. After the end of the 6-weeks training period, a test protocol identical to the baseline one was administered. The participants who received vitamin D supplements 30 days or less prior to blood sampling or suffered from acute respiratory viral infections or any other diseases that resulted in absence from three or more training sessions 30 days or less prior to the examination were excluded from the study. The players were advised to adhere to their usual dietary routines throughout the study and refrain from the consumption of any supplements or stimulants throughout the experiment. Additional eligibility criteria to participate in the study were outlined as follows: (a) a minimum of 10 years of training experience, (b) participation in a first-league team, (c) absence of injuries in the 6 months leading up to the assessments, and (d) consistent engagement in training sessions a minimum of 5 times per week over the last 6 months. All participants were informed verbally and in writing about the procedures, possible risks and benefits of the tests and provided written consent before the commencement of the study. All research procedures were reviewed and approved by the bioethical committee of the Academy of Physical Education in Katowice (ethic references KB-05/2017, December 5, 2017); furthermore, the study conformed to the tenets of the Declaration of Helsinki for medical research involving human subjects.

The experimental sessions were conducted between 8:00 and 10:00 a.m. The session was preceded by a warm-up protocol, which included 5 min of cycling, 5 min of treadmill running, several upper and lower body exercise with targeted DROM Exercises (Dynamic Range of Motion Exercise) (43), followed by 5 m and 30 m sprints, COD tests (90°) and two sets of running with submaximal speed. All sprint tests were performed on an indoor field with an artificial grass surface.

Twenty male elite soccer players from the First Polish League (age = 22.8 ± 8.2 years, body mass = 75.1 ± 15.3 kg, body height = 178.3 ± 13.5 cm, soccer training experience = 12 ± 3.4 years) took part in this study. The participants were all full-time professionals who trained daily.

The running times were recorded by two pairs of dual-beam Witty Gate photocells (Microgate, Bolzano, Italy). Following the warm-up phase, participants executed two successive 30-m sprints with a 5-min rest interval in between the trials (Figures 2A,B). To avert premature activation of the starting gate, participants commenced with their leading foot positioned 0.5 m behind the initial timing gate. The best time from the two trials, both at 5 and 30 m, was preserved for further analysis.

After the linear sprint test, participants rested for 5 min and then performed the COD tests (Figure 3). Each participant performed tests with a 90° COD (ZigZag test). The participant’s task’ was to cover a 30 m section with designated cones with changes in direction and proper movement pattern (44). The running test was performed twice with a 5-min rest interval between attempts. The fastest time from each COD test was retained for further analysis.

The COD deficit was calculated by the difference in run time in 30 m COD test [s] and 30 m linear sprint time.

Serum 25OH-Vitamin D was determined by RIA-CT KIP1971/KIP1974 (DIAsource ImmunoAssays SA, Louvain-la-Neuve, Belgium). For statistical analysis participants were divided into two groups according to the concentration of 25(OH)D: inadequate levels of 25(OH)D (vitamin D deficiency/insufficiency, <30 mg/mL), and adequate levels of 25(OH)D (vitamin D sufficiency, >30 mg/mL).

All statistical analyses were performed using Statistica 13.1. The Shapiro–Wilk test was used in order to verify the normality of the distribution. Pearson’s correlation and regression analysis were used to analyze the relationships between variables. Student’s t-test was used to verify the significance of differences for paired values. For pairwise comparisons, effect sizes were determined by Cohen’s d which was characterized as large (d > 0.8), moderate (d between 0.8 and 0.5), small (d between 0.49 and 0.20) and trivial (d < 0.05). Percent changes with 95% confidence intervals (95CI) were also calculated. Statistical significance was set at p < 0.05.

In the second aspect of our study, we investigated whether serum concentration of 25(OH)D affects speed and change of direction performance (COD) in professional soccer players. The main finding was that serum 25(OH)D concentration did not correlate with 5 m and 30 m linear sprint times, but it did correlate with COD performance. Comparisons between the level of 25(OH)D and Δ5 m [s] before-after and between 25(OH)D and Δ30 m [s] before-after did not reveal any significant associations. Significant correlations were found between the level of vitamin D and the effect of training on ΔCOD [s] before-after (p = 0.0035) (strong correlation) and Δdeficit [s] before-after (p = 0.039) (average correlation). In both cases, positive correlations were found, indicating that with an increase in the level of vitamin D, a significantly higher training effect was found. This resulted in a improvement of COD performance and a reduction in the deficit value. We also examined whether soccer players with a vitamin D level below 30 ng/mL (insufficient concentration) and those with a level above 30 ng/mL (sufficient concentration) experienced the same training effects and achieved similar results in the speed and COD tests. The level of 25(OH)D ≤ 30 or 25(OH)D > 30 did not significantly differentiate training effects for the variables Δ5 m [s] before-after and Δ30 m [s] before-after. However, a significantly higher training effect in the ΔCOD [s] before-after was found in soccer players with 25(OH)D > 30 compared to soccer players with 25(OH)D ≤ 30 (p = 0.0019). Also, a significantly higher training effect was found in case of Δdeficit [s] before-after in soccer players with 25(OH)D > 30 compared to soccer players with 25(OH)D ≤ 30 (p = 0.017). The results presented by other authors on the effect of vitamin D levels on performance are inconsistent. Most researchers studying soccer players confirm a positive correlation between vitamin D levels and speed and strength (20, 22, 27, 42, 45). In Gilic et al.’s (20) study with 52 young Croatian soccer players, better results were observed in speed tests over distances of 10 m and 20 m, as well as in COD tests in players with higher 25(OH)D. In our previous study (22) with 28 soccer players, we observed that the level of vitamin D at different times of the year influenced the results of speed tests over distances of 5 m and 30 m. Significant differences in 25(OH)D and 5 m speed test results were observed following summer compared with winter. Our results are consistent with the data presented by Jastrzebska et al. (42), Koundourakis et al. (45), and Książek et al. (30). In our research, similar to Jastrzebska et al. (42), soccer players with higher concentrations of 25(OH)D achieved better results in COD tests. They also achieved significantly better results on the deficit variable, which determines the differences in time over a distance of 30 m in a straight line and 30 m in a zig-zag manner. Unlike in our research, Jastrzebska and colleagues (42) found significant positive correlations between 25(OH)D concentration and sprint results over distances of 10 m and 30 m. The authors confirmed that players with a higher vitamin D level reached better results in the speed tests. They explained this by stating that the vitamin D resources stored in athletes’ bodies effectively influence the level of their anaerobic fitness. However, they concluded that it is still unclear whether the observed changes are due to differences in 25(OH)D concentration or applied training loads. In their opinion, the changes in speed and explosive power demonstrated in their study were caused by the applied training loads rather than by changes in 25(OH)D concentration. In turn, Skalska et al. (54) suggested that a higher 25(OH)D concentration with the same training load can effectively improve the level of anaerobic fitness. Also, Książek and colleagues (29) determined the relationship between 25-(OH)D concentration and performance in 24 soccer players. These authors showed a significant association between 3-epi-25-(OH)D3 and handgrip strength and vertical jump variables in soccer players and concluded that vitamin D metabolites might be involved in skeletal muscle function (18, 55). Contrary to the previous study, some authors did not confirm an association between vitamin D serum concentrations and strength performance in soccer players (5, 29). Książek et al. (29) did not find any correlations between 25(OH)D concentration and muscle strength or maximum oxygen uptake in Polish youth soccer players. Also, Branstrom et al. (5) in a study with Swedish female soccer players did not confirm a correlation between 25(OH)D and muscle performance assessed through isokinetic knee extension and flexion, countermovement jump, and sprint running.

Mechanisms by which vitamin D can influence speed performance are still hypothetical (20, 34, 36). Vitamin D controls and regulates the expression of muscle proteins and other cellular proteins synthesis, which are involved in calcium signaling and phosphate-dependent cellular metabolism, including energy resynthesis from ATP and phosphocreatine (34, 38, 56). Also, vitamin D influences muscle cellular calcium concentrations, which directly impact muscle contraction (19, 57). Vitamin D increases the influx of calcium into the cytoplasm by activating cellular kinases, empowering calcium to bind to the troponin-tropomyosin complex, resulting in exposure to active binding sites and allowing muscle contraction (20, 58). The more calcium ions are released into the cytoplasmic area, the more efficient the movement of myosin fibers through actin filaments, which may result in greater muscle contractile force and directly impact results in sprint and change of direction performance (9, 56). Additionally, VDR receptors present in myofibrils suggest a vitamin D genomic effect in their area by increasing the synthesis of muscle fibers (57). Vitamin D influences the size of fast-twitch fibers (37), which are important for the explosive type of human movement. Fast-twitch muscles can increase the muscle’s ability to generate energy very fast, resulting in higher speed and better neuromuscular coordination (20, 59). Vitamin D also increases the expression of IGF-1, which has an important role in muscle remodeling and hypertrophy (31). It is also worth adding that a higher level of vitamin D affects the synthesis of testosterone, which increases the synthesis of muscle proteins and indirectly affects muscle cellular metabolism and, finally, muscular speed and strength (9, 21, 37).

Due to the high use of vitamin D by athletes’ bodies every day, its level should be monitored throughout the year (17, 22). In the case of football players whose blood tests show a deficit or low level of 25(OH)D, it should be supplemented (32, 45). Our results confirmed previous studies that in soccers whose 25(OH)D level was above 30 ng/mL at the beginning of the preparatory period, training loads significantly improved speed (22, 32, 45). However, much research still needs to be done in this area to clearly confirm our results.

Our study has several limitations. Firstly, the absence of a control group impacts the robustness of our findings. Additionally, we did not measure dietary vitamin D intake and serum vitamin D concentration after the preparatory period. The study also did not assess other molecules such as 1.25(OH)D, Vitamin D Binding Protein (VDBP), or molecular variables such as vitamin D receptor (VDR) polymorphism or its mutations. These omissions limit the accuracy of interpreting potential mechanisms influencing vitamin D’s impact on performance. Furthermore, the study’s small sample size, limited to professional soccer players, restricts the generalizability of our results to other sports. Exploring the effects of various training stresses on vitamin D status could yield interesting insights but was not addressed in this study. In turn the strengths of our study include the research group, which consisted of professional Premier League soccers, and the project’s assumptions, which require minimal financial resources and are easy to implement by coaches in most soccer clubs. In the future, we plan to conduct similar studies with other groups of athletes, incorporating vitamin D supplementation.