The influence of cryostimulation on reducing inflammation and improving motor skills in football players

doi:10.21203/rs.3.rs-4959683/v1

Introduction:

Cryostimulation is one of the methods used for enchancing post-exercise recovery, involving short-term (3 min) exposure of the body to extremely low temperatures, oscillating between -100°C and -190°C. The purpose of this intervention is to reduce inflammation and alleviate physical as well as mental fatigue, which helps prepare the body for further exercise in the course of the training process.

Methods:

The study was conducted to determine the effect of five days of Partial-Body Criostimulation on inflammation and motor skills in soccer players. A group of 24 football players was randomly divided into a test group subject to cryostimulation (-140°C ± 20°C, 3 min, 5 days) and a control group. Before the cryostimulation session, both groups underwent visual-motor ability tests, which were repeated on the last day of the study. Blood samples were collected at four time points (P0 - 1^st day, P1 - 3^rd day, P2 - 5^th day, P3 - 2 days after the test). Levels of creatine kinase, TNFα, IL-6, IL-10, testosterone and cortisol were determined in the samples.

Results:

Analysis of the results revealed a significant improvement in motor function (reaction time, RT) in the experimental group by 2.43 seconds (p=0.001) compared to the control group. There was no significant difference between control and experimental groups regarding parameters determined in blood samples.

Conclusions:

The applied five-day cryostimulation regimen did not significantly affect the profile of inflammatory markers in soccer players. However, a reduction in visuo-motor reaction time (RT) was registered in cryostimulation-treated soccer players, suggesting the potential benefits of this method in improving motor skills.

Trial registry ClinicalTrials.gov ID: NCT06549933, retrospectively registered on 10.08.2024 .

cryostimulation

regeneration

football players

inflammation

motor functions

Among many recovery enhancing techniques systemic cryostimulation has been shown to have a positive effect on muscle recovery [1]. In addition, cold therapy is used in acute musculoskeletal injuries, chronic inflammation and optimization of recovery after exercise [2, 3]. Both WBC (Whole Body Cryostimulation) and PBC (Partial Body Cryostimulation) can be defined as exposure of the body to cold, dry air (-100°C to − 190°C) in a room called a cryochamber or cryosauna for a short period of time (2–3 min), cooled by liquid nitrogen vapor.Cryostimulation is usually used as a single application or in a cycle of several days during recovery periods [4, 5]. WBC/PBC cause increased activation of the adrenal sympathetic nervous system and the hypothalamic-pituitary-adrenal (HPA) axis in response to stress [6, 7]. Through interaction of the HPA axis, ANS (autonomic nervous system) and immune system, stress triggers multiple physiological responses, such as cortisol (C) secretion [6, 7, 8]. The relationship between exercise and C levels associated with cold stress is still under debate One study reported that in soccer and rugby players longer time is needed to sufficiently recover compared to other team sports disciplines, judging by the outcomes of physical endurance trials as well as CK and C levels [9]. Despite the somewhat inconsistent and inconclusive results, it appears that the exposure to cold temperatures positively impacts the athlete's performance and motor function. Suggested mechanism consists in counteracting the performance-reducing effects of high crtisol levels, by reducing the increases of this hormone.

Soccer is characterized by variable intensity of effort, including both low- and high-intensity activities, that can occasionally lead to fatigue when the athlete's physical and mental capabilities are exceeded [10, 11]. Intense exercise induces an increase in pro-inflammatory cytokines, such as tumor necrosis factor alpha (TNF-α) and interleukin-6 (IL-6). This process is offset by the release of the anti-inflammatory interleukin 10 (IL-10), [12].

The hormones testosterone (T) and C are important biomarkers for detecting changes in physical performance and exercise adaptation. T is associated with anabolism and muscle regeneration, while C, a stress hormone, is an indicator of catabolism and physiological load [13]. In addition, markers of muscle damage, such as creatine kinase (CK), are important indicators in assessment of the muscle damage and recovery after intense exercise [14].

The balance between these biomarkers plays a key role in monitoring the physiological state of athletes and in planning training and recovery strategies. The effectiveness of recovery methods, such as cryostimulation, can be evaluated through their effects on the levels of these biomarkers and on athletes' motor skills, as well as on overall physical performance. Research showed that fatigue is a potential factor influencing athletic performance by inducing changes in the neuromuscular and metabolic systems and modulating psychometric responses [15,8.11,16,9]. In the context of soccer, CK parameters and hormonal biomarkers appear to be particularly important as key indicators of the recovery process [17]. In open sports like soccer, players are required to quickly convert visual stimuli into a specific motor response (visual-motor response). Neuronal mechanisms potentially contribute to better motor response performance [18]. Reaction time (RT) and anticipatory ability are key aspects of perceptual abilities [19]. The use of modern electronic equipment allows to optimize the control of training effects. The following are cited as advantages of electronic systems: rapid test results, a choice of different testing options (individual, simple, complex), equipment mobility and comprehensive use in training of various sports [20, 21, 22, 23].

In recent years, there has been an increase in research on the effectiveness of cryostimulation, which has contributed to the popularization of WBC as a form of recovery in soccer [24]. However, detailed studies and data on inflammatory mediators and motor response time using cryogenic temperatures are very limited and insufficient to assess the true potential of this method. Due to the controversy over the effectiveness of WBC and PBC in biological recovery, our study aimed to evaluate the impact of the cold factor on the athlete’s recovery. We hypothesized that five-day PBC applied once a day will speed up the recovery process and improve motor (visual-motor) abilities in soccer players. The aim of the study was therefore to determine the effect of a 5-day PBC on reducing inflammation and improving motor skills in soccer players during the initial phase of the training period.

2.1 Participants

The participants of the study were soccer players (3rd league) of Solar Home Stilon Gorzow Wielkopolski club (n = 24). The players (age 21 ± 4.9 years, height 181.7 ± 7.7 cm, weight 75.6 ± 8.8 kg, BMI 22.8 ± 1.7), were at a similar level of training, with at least 10 years of training experience. Participants were randomly assigned to two groups: a partial cryostimulation group (PBC, n = 11) and a control group (CON, n = 10) (Fig. 1).

During the study, the participants did not use any other forms of recovery, cosmetic treatments, dietary supplements, or consume alcohol or other stimulants. In addition, the football players had no previous experience with systemic cryotherapy before participating in the study. The exclusion of other forms of regeneration and stimulants was intended to eliminate confounding variables, allowing a more precise determination of the effect of cryostimulation on the physiological and motor parameters analyzed.

2.2 Design

Prior to the application of cryostimulation, both PBC and CON groups underwent anthropometric measurements using a Tanita BC-418 MA analyzer (Europe GmbH, Sindelfingen, Germany) and elbow vein blood samples were taken to determine hormone levels (C, T) and inflammatory markers (CK, IL-6, IL-10, TNFα) at four time points. In addition, motor performance tests (reaction time, RT) were conducted using the Witty Sem semaphore system (Microgate SRL, Bolzano, Italy).

The athletes in the PBC group were subjected to daily cryostimulation in a JUKA 0104-1 cryosauna (Jett, Nurnberg, Germany). The main part of the study consisted of five days of cryostimulation (PBC), which was applied daily between 8:00 and 9:00 a.m., lasting 3 minutes, and moderate-intensity training between 4:30 and 6:00 p.m. The experiment was conducted 48 hours after the end of a league match, which took place in harsh weather conditions characterized by air temperature of -4°C, frozen turf and snowfall.

Control:

On the last day of the experiment, control tests were conducted again and further blood samples were taken, as shown in Fig. 1.

2.3 Procedures

2.3.1 Intervention (PBC)

During the cryostimulation session, which took place daily between 8:00 and 9:00 a.m. for 5 days, participants in the PBC group were placed in a cryosauna for a period of 3 minutes, where the temperature was − 140°C ± -20°C, achieved by cooling with liquid nitrogen. The cabin was equipped with a cover placed at shoulder height and covering the head to protect participants from direct contact with the extremely low temperature. Participants entered the chamber wearing clogs, wool socks, shorts, gloves, caps and disposable masks made of non-woven PP fabric to provide protection from inhaling nitrogen vapors. Before the start of the cryostimulation session, all participants received instruction on proper breathing and behavior during the therapy. During the treatment, the athletes remained in constant visual and auditory contact with the therapy supervisor to ensure safety and comfort during the procedure.

Participants were informed of the voluntary nature of participation in the study and that they could opt out at any time, in accordance with the principles of the Declaration of Helsinki. Before taking part in the study, each participant signed a written informed consent and presented a certificate from a sports doctor confirming that there were no contraindications to systemic cryotherapy. The study was approved by the Bioethics Committee at the Karol Marcinkowski Medical University in Poznan (Resolution No. 845/22) and registered in the Clinical Trials Registry (ANZCTR of Australia and New Zealand, ACTRN; reference no. 12622001541796).

2.3.2 Study group characteristics

Body mass index (BMI), total body water (TBW), fat mass (FM), fat-free mass (FFM), and basal metabolic rate (BMR) were measured on an empty stomach in two stages: the first day of testing before the cryosauna treatments and the fifth day after the last PBC session. The device was calibrated before each testing session according to the manufacturer's instructions. Height and weight measurements of soccer players were taken (Table 1).

Table 1

Anthropometric characteristics of participants
	CON n = 10	PBC n = 11
Height [cm]	182,7 ± 6,58	178,9 ± 6.9
Weight [kg]	76,69 ± 8,91	72.46 ± 7.61
FM [kg]	9,59 ± 1,70	9,35 ± 2,53
FFM [kg]	67,11 ± 9,18	63,11 ± 7,54
TBW [kg]	49,15 ± 6,72	46 ± 5,52
BMI [kg/m^2]	22,93 ± 1,94	22,62 ± 1,63

*CON control group; PBC, Partial-Body-Cryostymulation group; BMI body mass index; FM fat mass; FFM fat free mass; TBW total body water.

2.3.3 Motor test (response time RT)

Motor tests, measuring response time (RT), were conducted using a semaphore system consisting of an LED array equipped with proximity sensors. These sensors recorded movement by capturing a beam of light, making it possible to perform the test without touching the light screen (an area of 40 cm maximum). The 209-cm-wide rack was equipped with four semaphores spaced 49 cm apart one from the other, arranged in a wave configuration. The software used was AGILITY, which assessed peripheral vision, reaction time, decision-making and eye-hand coordination. The external sensors were placed at a height of 156 cm, and the internal sensors were placed at a height of 172 cm from the ground. The system analyzed only correct decisions, based on hand proximity. The test subject stood at an extended upper limb distance from the semaphores. The start of the test was signaled by a sound and light on the semaphores. The subject's task was to locate 20 letters "a" in green, having unlimited time to do so, which was measured and served as an indicator of response to a light (visual-motor) stimulus. Due to the specific nature of the game of soccer, i.e. the execution of fast and precise movements (turns, jumps, slides) of the body in response to a visual stimulus, the test was not required to be performed with the dominant limb. Time measurements, made to the nearest ± 0.4 ms, were made in seconds for both groups on both the first and fifth day of the study. Before the start of the study, validation of the device was performed to eliminate the learning effect. During the 10 trials (n = 24), conducted at 5-minute intervals, the average time result was 1.4 seconds.

2.3.4 Blood sampling

Venous blood sampling was conducted in four stages: P0 before the start of PBC therapy, P1 on the third day of therapy, P2 after five sessions, and P3 two days after the end of the study to assess the long-term effect. Blood samples were collected in the morning (8:00–8:30 am) from the ulnar vein, using S-Monovette vacuum syringes. The samples were then subjected to centrifugation at 3,000 rpm, at 4°C for 10 minutes, in the laboratory of AWF Poznań, Gorzów Wielkopolski branch. The material was stored at -80°C until assays were performed. Serum levels of biochemical indicators of fatigue and inflammation were determined. Concentrations were measured: tumor necrosis factor (TNF-α, sensitivity 2.827 ng/L), interleukin IL-6 (sensitivity 0.857 ng/L), IL-10 (sensitivity 9.012 ng/ml), total creatine kinase activity (CK, sensitivity 0.746 ng/L), cortisol (C, sensitivity 2.42 ng/L) and testosterone (T, sensitivity 0.10 ng/L), using ELISA Kit (Dia Metra S. r.l, Spello, Italy), using the "double-antibody sandwich" technique.

Statistical analyses were performed using the R language and a dedicated statistical computing environment [25]. To assess the repeatability of functional test execution time measurements, the mean standard deviation (SD) of a series of individual measurements was calculated. Before selecting the method of analysis, the normality of the distribution of individual variables was assessed using the Shapiro-Wilk test. Changes over time in the performance of the functional tests were estimated as differences in their nominal values (∆tafter - ∆tbefore), and also as percent relative changes (RC) based on measurements taken before (∆tbefore) and after (∆tafter) the cold exposure cycle, according to the respective formulas.

RC = ln((Δt_after):(Δt_before ))["%"].

The change values were developed by calculating the average values of the indicator sets for the control and test groups, as well as the average differences between the groups. Cohen's d effect size indices and the probability of zero change values were calculated using Welch's t-test. Non-parametric methods were used to estimate the effect size of changes in biochemical indices. The study used Cliff's index δ [26], as an effect size measure describing the monotonicity of value changes in repeated measurements of individual indicators and their relationships. The probability of zero change was evaluated using the Wilcoxon test.

4.1 Biochemical parameters

Analysis of tested hormonal, immunological and biochemical indices yielded no statistically significant results, however certain differences and trends were noted. A decrease in C levels was observed in both the control group (111 ng/ml) and the test group (108 ng/ml) on the second day of the experiment. The PBC group maintained a marked decrease in C levels until the end of the study (P0-141 ng/ml, P1-108 ng/ml, P2-122 ng/ml, P3-125.5 ng/ml), while in the control group (CON) the decrease was smaller and remained at similar levels (P0-143 ng/ml, P1-111 ng/ml, P2-133 ng/ml, P3-132 ng/ml), as shown in Fig. 3. A similar effect was observed in CK levels in the PBC group, where CK levels decreased on the second day of the study (P0-58.6 ng/ml, P1-53.4 ng/ml, P2-53.3 ng/ml) and persisted until the fifth day. In the CON group, CK levels increased on the second day of the study and remained constant until the fifth day (P0-51.9 ng/ml, P1-55.1 ng/ml, P2-55.6 ng/ml), as shown in Fig. 4. Two days after the study, C levels in the PBC group were still lower (P4-125.5 ng/ml) compared to the CON group (P3-132 ng/ml). CK levels in the PBC group two days after cryostimulation returned to baseline (P0-58.6 ng/ml, P4-58.2 ng/ml), while those in the CON group increased compared to baseline (P0-51.9 ng/ml, P3-61.8 ng/ml). Testosterone/cortisol ratio (T/C) and levels of other markers (IL-10, IL-6, TNFα) 48 hours after the match showed no change.

Neither the values of the Cliff effect (0 ± 0.147 negligible, 0.147 ± 0.33 small, 0.33 ± 0.474 medium, 0.474 ± 1 strong) size nor the probability of equality thesis of the values of the biochemical indices authorise the assumption of any effect of the applied thermal stimulus on changes in the biochemical indices analysed.

4.2 Motor test

The average of standard deviations determining the repeatability of visual-motor reaction time measurements was 1.64s (1.47s,1.8s). After eliminating the learning effect that we observed in the measurement data, this indicator took the value of 1.4s (1.19s, 1.6s). The test completion time decreased in both the PBC ( 22.26s to 18.65s) and CON (21.16s to 19.99s) groups (Table 2). However, the decrease in the PBC group was much more pronounced, with values 2.43s smaller than in CON group (p = 0.001, d = 1.38), as shown in Fig. 5. Cohen's effect size interpretation was adopted: d < = 0.2 - trivia, d = 0.2 ÷ 0.5 -small, d = 0.5 ÷ 0.8 - medium, d > 0.8 - large.

Table 2

Group reaction time before and after a cycle of PBC
	Mean values [s]		Nominal differences			Relative change
Group	before	after	mean [s]	d	p	mean [%]	d	p
CON	21.16 [19.73, 22.59]	19.99 [18.27, 21.7]	−1.17 [− 2.26, − 0.082]	−0.77 [− 1.48, − 0.053]	0.038	−5.97 [− 11.5, − 0.44]	−0.77 [− 1.49, − 0.057]	0.037
PBC	22.26 [20.02, 24.50]	18.65 [17.34, 19.96]	−3.60 [− 5.36, − 1.85]	−1.38 [− 2.05, − 0.71]	0.001	−17.1 [− 25.2, − 8.98]	−1.41 [− 2.08, − 0.74]	0.001
PBC/CON			−2.43 [− 4.38, − 0.48]	−1.15 [− 2.08, − 0.23]	0.018	−11.1 [− 20.4, − 1.88]	−1.11 [− 2.03, − 0.19]	0.021

After the application of cryostimulation in the presented study, changes were observed in the speed of motor agility (visual-motor response) in football players in response to a light stimulus (p = 0.001, d = 1.41). The players subjected to PBC reacted faster to the light signal (green letter "a"). Previous research suggests that a decrease in natural body temperature after cold exposure can negatively affect cognitive-motor function [8, 27], although most reports have assessed cognitive performance during or after cold water immersion (10°C), [27, 28].

Our study is one of the first to analyze the effects of PBC on the motor skills (reaction speed, RT) of football players using modern electronic technologies. A possible explanation for the changes in motor function may be the effect of cryostimulation on brain-derived neurotrophic factor (BDNF), which increases after exercise (after one session). BDNF improves cognitive function and can modify glucose and fat levels [29]. Jaworska's research [30] showed an increase in BDNF levels after exposure to whole-body cryotherapy (-110°C, 3 min, 10 times) in volleyball/volleyball players (n = 8), which contributed to an improvement in motor skills (concentration and accuracy of the motor task) in a photobucket test (SMART SPEED PRO-Fusion Sport).

The results indicate that the recovery process of the football players assisted by PBC did not result in changes in inflammatory markers (Fig. 3,4) We expected that the group subjected to PBC, under the influence of cold factor (stressor), would show increased activation of the hypothalamic-pituitary-adrenal (HPA) axis, which is a typical response to stress [6, 5]. However, the observed hormonal profile remained at a steady, declining level.

An effect similar to that observed in our study had been noted after a single exposure to whole-body cryotherapy (WBC, -135°C, 3 min), where there were no significant differences in CK, C and T/C in a group of football players (n = 14) previously subjected to sprint exercise (15 × 30 m) [31]. Nevertheless, elevated T levels were noted 24 hours after the WBC, which had no effect on other measured parameters. Similar increases in T levels were noted after an elite rugby league match (n = 23) in players subjected to two subsequent WBC exposures (-120°C to -135°C, two sessions 3 min each) [32]. An increase in the T/C ratio and a statistical trend of CK reduction was observed 60 hours after double WBC exposure, while similar effect was not observed after one 3 min WBC exposure. These findings suggest that WBC immediately after exhaustive competition may stimulate the anabolic profile, and that this effect depends to some extent on the exposure time.

Similar results regarding the decrease in C after two sessions of PBC were reported in a study where rugby players (n = 25) were subjected to PBC (-110°/-140°C, 3 min) twice a day for 7 days [33]. In our study, C decreased after two exposures, but returned to its initial value after five PBC sessions. An interesting modulation of C and T levels was obtained in a study, using a five-day PBC (-120°C, 3 min, twice a day) in a group of tennis players (n = 12) undergoing moderate training efforts [34]. C and T values corresponded to physiological reference values, but showed some modulation. The authors concluded that cryostimulation can support a training program. It should be emphasized that both in the mentioned study [34] and in our C and T values did not exceed the reference ranges.

The results of our own study, as well as those of other authors [6, 7, 8, 33, 34, 9], show that partial body cryotherapy (PBC) has a multidirectional effect on the muscular and nervous systems, so its application requires precise planning, taking into account the specifics of the sports discipline and the training load. The stimulating effect depends on the number of WBC or PBC exposures applied, as well as the cooling temperature. Cryotherapy can be particularly beneficial in sports where quick reactions and precise, coordinated movements are crucial, such as football and rugby.

A limitation of our study was the small size of the study group (n = 24). However, given the relative homogeneity of the participants in terms of age, training history and type of sport, the sample size was considered sufficient to conduct the experiment and draw reliable conclusions. Three participants dropped out of the study due to injury (1) and upper respiratory illness (2).

The study protocol did not include a blinded control trial (CON), mainly due to difficulties in masking the temperature of partial cryotherapy. Information about the preparation, feelings and potential risks of PBC was provided to participants in advance, which may have affected motor function. Therefore, future studies should consider blinding the trial to minimize the impact of this factor on the results. Research is needed that determines the effectiveness of combinations of PBC interventions from a motor-cognitive ability perspective using innovative electronic systems.

The findings of our study indicate potential benefits of cryostimulation in improving motor function and recovery in athletes, but further exploration is required to fully understand the mechanisms and optimal conditions for using this method. Developments in technology and innovative approaches to monitoring the effects of cryotherapy may contribute to a better understanding of its effects on the human body and to optimizing recovery strategies in sports.

Five days of cryostimulation showed no significant effect on the levels of inflammatory markers in soccer players during the period of detraining. Judging from statistical trends of CK and C levels, PBC appears to protect against muscle damage, however changes and differences between PBC and control group were not significant in that matter. An improvement in motor (visuomotor) function was observed in soccer players subjected to PBC.

PBC has the potential to support training, increasing its effectiveness by reaction time reduction. PBC appears to be a safe method of reducing exercise-induced damage, allowing its use during periods of recovery and regular training. However, it should be considered as a complement to other forms of recovery, and not as the only procedure used for this purpose.

PBC- Partial-Body Criostimulation

WBC-Whole-Body-Criostimulation

HPA- hypothalamic-pituitary-adrenal

ANS- autonomic nervous system

CK- creatine kinase

C-cortisol

T-testosterone

TNF-α- such as tumor necrosis factor alpha

IL-6- interleukin-6

IL-10- interleukin-10

RT- response time

7. Electronic supplementary material.

8. Funding

No funding. Own contribution.

9. Conflict of interest

The authors declare no conflict of interest.

10. Availability of data and materials

The datasets generated and/or analyzed during the study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate

The study was approved by the Bioethics Committee at the Karol Marcinkowski Medical University in Poznan (Resolution No. 845/22) and registered in the Clinical Trials Registry (ANZCTR of Australia and New Zealand, ACTRN; reference no. 12622001541796).

Consent for publication

Not applicable.

Competing interests

The authors have no relevant financial or non-financial interests to disclose.

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No competing interests reported.

The influence of cryostimulation on reducing inflammation and improving motor skills in football players

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Version 1

Abstract

Figures

1. Introduction

2. Materials and methods

2.1 Participants

2.2 Design

2.3 Procedures

2.3.1 Intervention (PBC)

2.3.2 Study group characteristics

2.3.3 Motor test (response time RT)

2.3.4 Blood sampling

3. Statistical analysis

4. Results

4.1 Biochemical parameters

4.2 Motor test

5. Discussion

6. Conclusions

Abbreviations

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1