Moderate-dose caffeine enhances anaerobic performance without altering hydration status

Frontiers in Nutrition 01 frontiersin.org

Moderate-dose caeine

enhances anaerobic performance

without altering hydration status

AhmetMor

, KürşatAcar

, DanIulianAlexe

*, HakkıMor

MekkiAbdioğlu

, MariaCristinaMan

*, FatihKarakaș

FatmaBenWaer

, AliKerimYılmaz

and CristinaIoanaAlexe

Department of Coaching Education, Faculty of Sport Sciences, Sinop University, Sinop, Türkiye,

Department of Physical Education and Sports, Faculty of Sport Sciences, Sinop University, Sinop,

Türkiye,

Department of Physical and Occupational Therapy, “Vasile Alecsandri” University of Bacau,

Bacau, Romania,

Department of Coaching Education, Yasar Doğu Faculty of Sport Sciences,

Ondokuz Mayıs University, Samsun, Türkiye,

Faculty of Sport Sciences, Institute of Health Sciences,

Ankara University, Ankara, Türkiye,

Department of Physical Education, 1 Decembrie 1918 University,

Alba Iulia, Romania,

Research Laboratory Education, Motricité, Sport et Santé (EM2S) LR19JS01, High

Institute of Sport and Physical Education of Sfax, University of Sfax, Sfax, Tunisia,

Recreation

Department, Yasar Doğu Faculty of Sport Sciences, Ondokuz Mayıs University, Samsun, Türkiye,

Department of Physical Education and Sports Performance, “Vasile Alecsandri” University of Bacau,

Bacau, Romania

The eects of direct nutritional supplements on athletic performance are still

being investigated and arouse curiosity. Only one study in the literature was

found that investigated the kicking speed performance of futsal players following

low-dose caeine supplementation (3 mg/kg); thus, the question of whether

caeine supplementation improves kicking speed as well as essential physical

parameters in soccer players is still controversial. Therefore, the aim of this

study was to determine the eect of caeine supplementation on vertical jump

(VJ), sprint, reaction time, balance, change of direction (COD), and ball-kicking

speed in soccer players. In a double-blind, cross-over design, nine moderately

trained male soccer players (21.11 ± 2.02 years, 171.22 ± 6.14 cm, 71.78 ± 10.02 kg)

consumed caeine (6 mg/kg) or a placebo 60 min before completing balance,

reaction time, vertical jump, agility, 30 m sprint, and ball-kicking speed tests.

Greater VJ height (p = 0.01) and power (p = 0.08), and faster completion time

according to the Illinois Agility Test (p = 0.08) were found following caeine

supplementation compared to placebo. Elapsed time (p = 0.01), average

(p = 0.01) time, and the slowest reaction times (p = 0.016) were signiﬁcantly

reduced after caeine consumption compared to placebo supplementation.

Caeine intake signiﬁcantly improved VJ, agility, and reaction time (p < 0.05) but

did not aect 30 m sprint, ball-kicking speed, balance, and RPE values in soccer

players (p > 0.05). Although non-signiﬁcant, caeine intake also improved

sprint (0.67%) and ball kicking (2.7%) performance percentages. Also, caeine

consumption did not induce dehydration, and the athletes’ body hydration

levels were normal. These ﬁndings support the use of caeine supplementation

as an eective nutritional ergogenic aid to enhance anaerobic performance, at

least for vertical jumps, COD speed, and reaction time, in trained male soccer

players.

KEYWORDS

sports nutrition, supplements, ergogenic aid, soccer, caeine

OPEN ACCESS

EDITED BY

Marios Hadjicharalambous,

University of Nicosia, Cyprus

REVIEWED BY

Alvaro López Samanes,

Comillas Pontiﬁcal University, Spain

Fahri Safa Cinarli,

Inonu University, Türkiye

Eleanna Chalari,

Aegean College, Greece

*CORRESPONDENCE

Dan Iulian Alexe

[email protected]

Maria Cristina Man

[email protected]

RECEIVED 22 December 2023

ACCEPTED 26 June 2024

PUBLISHED 09 July 2024

CITATION

Mor A, Acar K, Alexe DI, Mor H, Abdioğlu M,

Man MC, Karakaș F, Waer FB, Yılmaz AK and

Alexe CI (2024) Moderate-dose caeine

enhances anaerobic performance without

altering hydration status.

Front. Nutr. 11:1359999.

doi: 10.3389/fnut.2024.1359999

Karakaș, Waer, Yılmaz and Alexe. This is an

open-access article distributed under the

terms of the Creative Commons Attribution

License (CC BY). The use, distribution or

reproduction in other forums is permitted,

provided the original author(s) and the

original publication in this journal is cited, in

accordance with accepted academic

practice. No use, distribution or reproduction

is permitted which does not comply with

these terms.

TYPE Original Research

PUBLISHED 09 July 2024

DOI 10.3389/fnut.2024.1359999

Mor et al. 10.3389/fnut.2024.1359999

Frontiers in Nutrition 02 frontiersin.org

1 Introduction

Soccer performance is characterized by bursts of high-intensity

physical activity, which requires players to simultaneously perform

intense running, and explosive soccer-specic actions such as kicking,

jumping, sprinting, and agility (1). An ergogenic aid is dened as any

intervention encompassing training techniques, mechanical devices,

nutritional components, pharmacological methods, or psychological

strategies that can enhance exercise performance capacity or improve

training adaptations (2). us, the ecacy of ergogenic aids, such as

caeine, on anaerobic performance is of interest to soccer players,

coaches, and sports scientists. Caeine is the most widely used

ergogenic aid by the athletic population (3). Indeed, following the

removal of caeine from the World Anti-Doping Agency’s list of

banned substances, it has been reported that three of every four

professional athletes use caeine before or during competition (4). As

such, in a study by Tallis etal. (5) 35 of the 36 clubs across the English

professional soccer leagues reported that caeine was administered to

enhance soccer performance. e rationale behind this phenomenon

is the underlying mechanisms by which the ergogenicity of caeine

may benet anaerobic performance, which are the antagonism of the

adenosine receptor at concentrations in the micromolar range in the

central nervous system (CNS) (6), increased β-endorphins secretion

via activation of the hypothalamic-pituitary-adrenal (HPA) axis (7),

and intracellular calcium release from the sarcoplasmic reticulum in

muscle cells (8), all of which can benet performance via improving

power, agility, reaction time, and alertness and delaying fatigue (6).

e ergogenic eect of caeine supplementation has been

reported in a variety of soccer-specic performance parameters, such

as improved performance in the Loughborough Soccer Passing Test

(9), the 20 m sprint test (10), fatigue resistance in 75% of the VO2max

to volitional fatigue test (11), jumping performance in the

countermovement jump (CMJ) test (11, 12), agility in the arrowhead

agility test (10), and reaction time (10, 11, 13). However, direct

extrapolation of these ndings to the complexity of soccer is

complicated, and no study has yet investigated all these parameters in

the same test setting with the same participant groups (6). Since the

ergogenicity of caeine may beinuenced by various factors, such as

genotype (14), training status (15), habituation to caeine (16), and

supplementation regimen, various physical performance responses to

caeine can dier even in the same person (6). erefore, evaluating

the eect of caeine supplementation on all these soccer-specic skills

and/or parameters in the same eld test battery is important for

improving understanding of the potential of caeine supplementation

to soccer performance.

Kicking is another key skill and/or component of soccer. Indeed,

ball-kicking speed has been suggested as a new, ecient performance

indicator in youth soccer players (17). Given that the ball-kicking

speed is associated with and/or aected by various physical aspects

(e.g., technique, power, and balance), it might beanticipated that

caeine supplementation has the potential to inuence ball-kicking

speed; however, empirical evidence to support this is presently lacking.

While one previous study by Lopez-Samanes etal. (18) reported no

dierence in ball velocity performance in futsal players aer low-dose

of caeine (3 mg/kg) supplementation, it remains unknown whether

caeine supplementation improves the ball-kicking speed in soccer

players. As such, further research is required to assess the eect of

caeine supplementation on the ball-kicking speed in soccer players.

erefore, the aim of this study was to determine the eect of

moderate-dose (6 mg/kg) caeine supplementation on vertical jump,

sprint, reaction time, balance, agility, and ball-kicking speed in the

same eld test battery in moderately trained male soccer players. In

addition, body hydration levels and uid balance were analyzed to

determine the eect of caeine on body water homeostasis. It was

hypothesized that (I) the ingestion of moderate-dose of caeine would

improve vertical jump, sprinting, reaction time, balance, agility, and

ball-kicking speed and (II) not induce dehydration and

uid imbalances.

2 Materials and methods

2.1 Participants

Nine healthy, non-smoking, young, moderately trained (19),

male soccer players participated in this study (age 21.11 ± 2.02 years,

height 171.22 ± 6.14 cm, weight 71.78 ± 10.02, habitual consumption

of caeine 188 ± 83 mg d

−1

; mean ± SD, Table1). e required sample

size was estimated using G*Power soware (Heinrich-Heine-

University Düsseldorf, version 3.1.m9.2, Düsseldorf, Germany). A

sample size of eight participants was determined sucient (eect

size: 0.50, condence interval: 1-β 0.95, error: α 0.05, actual power:

0.96). Based on Acar etal. (20) and discussion between the authors,

weset the eect size at 0.5. All participants had 9.89 ± 2.57 years of

competitive soccer experience at club standard and at least 3 years of

experience playing in regional and university-level leagues.

Participants completed at least ve weekly soccer training sessions

(7.5 h a week). All participants declared that they had not used any

ergogenic aids that might alter body hydration levels and exercise

performance within 3 months from the start of the study. All

participants were informed of the experimental procedures before

giving their written informed consent.

2.2 Experimental design

On the rst visit, a habitual caeine consumption questionnaire

was given to the participants in addition to their anthropometric and

body composition assessments. Body mass was obtained in kg with a

bioelectric impedance analysis device (BIA, Inbody 120, InBody Co.,

Ltd. Seoul, Korea), and height was obtained in cm with a portable

stadiometer (Seca 213, Hamburg, Germany). Habitual caeine intake

was determined through a validated questionnaire (21). Only

participants with a daily caeine intake of less than 250 mg d

−1

were

included to control individual dierences in responsiveness to caeine

TABLE1 Descriptive information of subjects (n = 9).

Variables X SD

Age (yr) 21,11 2,02

Height (cm) 171,2 6,14

Weight (kg) 71,78 10

BMI (kg/m

) 24,37 1,9

Training age (yr) 9,89 2,57

Habitual consumption of

caeine (mg/day

−1

)

188 83

Mor et al. 10.3389/fnut.2024.1359999

Frontiers in Nutrition 03 frontiersin.org

from habituation. In addition, participants were selected based on the

following inclusion criteria: (1) were aged between 18 and 25 years; (2)

had actively participated in soccer training for at least 3 years, with a

minimum of three times/week for the last 3 three months; (3) were

non-smokers. On the rst visit, participants also performed the testing

protocol for familiarization at a low intensity that would not make

them exert vigorous eort. Following completion of this initial

familiarization, participants were assigned to ingest either caeine or

a placebo in a double-blind, cross-over, randomized counterbalanced

design. e participants were randomly assigned to the two conditions

using the Research Randomizer soware (www.randomizer.org;

accessed on 10 June 2022). Participants consumed 6 mg/kg of caeine

(Nature’s Supreme, Istanbul, Turkey) or placebo supplements (wheat

bran) in capsules (same color and form, made up of gelatin hard form)

60 min before the testing protocol (5). A researcher, who had no

further involvement in this research, prepared the caeine dose using

electronic laboratory scales with one milligram of sensitivity at room

temperature. ere were at least 48 h between sessions to ensure that

the caeine had washout and to allow participants to complete

recovery. Participants were asked to record their diet 24 h before the

rst test session and replicate it 24 h before the second test session. For

24 h before and for each of the testing sessions, participants were asked

to refrain from ingestion of caeine, ergogenic aids (e.g., nitrate,

sodium bicarbonate), alcohol, and anti-inammatory drugs; not to

engage in strenuous physical activity; and to be strict with their

nutrition and rest. Regarding the rhythm (22), the tests and

measurements were applied to the participants at the same time of the

day (between 1–3 pm), under similar environmental conditions

(ambient temperature 22.00 ± 1.41°C, humidity 62.00 ± 4.24%,

pressure 1018.00 ± 1.41 mbar; mean ± SD) in the Sinop University

indoor sports hall and performance laboratory. Participants were

instructed to wear the same clothing and footwear for all the testing

sessions. In the second and third sessions, participants’ hydration

levels were analyzed just before placebo or caeine ingestion and

immediately aer tests. Participants completed a 15 min standardized

warm-up. Ad libitum water consumption (similar amounts) was

allowed in both trials. e testing protocol in each experimental

session consisted of balance, vertical jump, reaction time, change-of-

direction via the Illinois Agility Test, 30 m sprint, and the ball-kicking

speed tests, respectively. Vertical jump, reaction time, change of

direction, 30 m sprint, and ball-kicking speed tests were performed in

the indoor sports hall while balance, hydration levels, and RPE tests

were conducted in the performance laboratory. ese performance

tests were employed because they were the same as soccer players’

moves in training and competitions. A 3 min passive rest period was

given between the performance tests (except the vertical jump test) to

facilitate recovery (23) and participants were allowed two trials with

the best score used for subsequent analysis (24). In addition, the Borg

CR10 scale with a range of 0–10 was used to measure the rate of

perceived exertion (RPE) at the end of the tests (25). A schematic

diagram of the experimental protocol is displayed in Figure1.

2.3 Anthropometric and body composition

assessments

Body mass was obtained in kg with a bioelectric impedance

analysis device (BIA, Inbody 120, InBody Co., Ltd. Seoul, Korea), and

stature was obtained in cm with a portable stadiometer (Seca 213,

Hamburg, Germany).

2.4 Balance test

A portable dynamic balance device (Togu Challenge Disc 2.0,

Prien amChiemsee, Rosenheim, Germany) was utilized to assess the

balance of the participants. e platform was free to move in all

directions (up to a maximum of 12°) and thus provided an unstable

ground. e challenge disc recorded the athlete’s movements with

three-dimensional motion sensors and sent the data in real time to its

soware on the smartphone or tablet via Bluetooth. Stability index

ranges were categorized into 1 to 5 (1—very good, 2—good, 3—

normal, 4—weak, 5—very weak), and a lower score (p) indicated a

better balance. Initially, the researcher showed the application on the

tablet to the athlete at eye level, and the athlete stood barefoot on the

platform to eliminate the possible eects of dierent types of shoes on

the results. Later, the athletes were instructed to stand in the middle

of the disc and keep their balance for 20 s (aer 10 s of preparation, 5 s

of which is a countdown) with their arms free to swing. During the

test, participants were told to keep the point in the circle as central and

stable as possible. e platform provided a safe measurement for

athletes with its non-slip surface. e test was performed two times

with a 3 minute passive rest, and the best score was used as the

dynamic balance test score (26).

2.5 Reaction time test

Participants’ reaction times were determined using the Light

Trainer (Reaction Development and Exercise System, Istanbul,

Turkey). e reaction time test course consisted of four light modules

(stuck to the top of 30 cm height trac training cones) lined up side

by side at a distance of 1 m. e test course was designed depending

on the facility conditions (indoor sports hall) and the physical

characteristics of the experimental group (trial with dierent numbers

of modules and feedback). Athletes stood 1 m from the modules in the

middle of the course. Participants got ready for the reaction time test

on the “ready” command. e athletes deactivated the light modules,

which were lit on the right or le side, with precise movements,

touching with their dominant hand to the top of the module. e test

started with activating the rst light automatically and ended with the

athletes’ “deactivating” the last light. Athletes were asked to deactivate

30 light modules in total. e test was repeated if the participants hit

the modules, dropped them, or deformed the test course. e test was

performed two times with a 3 minute passive rest, and the best score

was used as the reaction time test score (27).

2.6 Vertical jump and anaerobic power test

A digital vertical jump device (Takei 5,406 Jump-MD Vertical

Jumpmeter, Tokyo, Japan) was used to measure the vertical jump

scores of the participants. Firstly, the rubber vertical jump plate was

placed on a at surface. In order to eliminate the possible eects of

dierent types of shoes on the results, the participants were instructed

to take o their shoes and stand “ready” with bare feet centered on the

Mor et al. 10.3389/fnut.2024.1359999

Frontiers in Nutrition 04 frontiersin.org

plate (10–20 cm from each other). Aerward, the researcher (the same

person fastened the digital belt in all trials for test reliability) zeroed

the digital belt, wound it tightly around to the waist of the participants,

and turned the pulley gently in the direction of the arrow to take the

slack out of the rope. Once the athletes were ready, they quickly moved

from the upright standing position to a position of 90° exion of the

knees with freely swinging arms and jumped for the maximum height.

e test was repeated if participants jumped by stepping forward, the

measuring tape was loose, or they did not land on the rubber plate

aer jumping. Each player performed two trials interspersed with

1 min rest between each vertical jump, and the best (highest) jump was

recorded in cm with an accuracy of ±1 (28). Participants’ anaerobic

power calculations were executed using the Lewis formula: anaerobic

power (W) = {√4.9 [body weight (kg)] √vertical jump (m)} (29).

2.7 Illinois agility test

e COD was assessed with the Illinois Agility Test by using a

photocell (Seven, SE-165 Photocell Stopwatch, Istanbul, Turkey). e

agility course is an area of 10 m long and 5 m wide, formed by four

30 cm height trac training cones lined up in a straight line at 3.3 m

intervals from each other in the middle. e test consists of a 40 m

straight, and a 20 m slalom run with 180° turns every 10 m. e

photocell timing gates were placed at the start and nish lines at a

height of approximately 1 m. When they were ready, athletes started

the test 30 cm behind the starting point. e participants were asked

to run at maximal speed. e test was repeated two times with a

3 minute passive rest between each trial, and the best value was

recorded as the Illinois Agility Test time (30).

2.8 30M sprint test

e linear sprint times of the soccer players were determined with

the 30 meter sprint test by using a photocell (±0.01 s precision) device

(Seven, SE-165 Photocell Stopwatch, Istanbul, Turkey) in the indoor

sports hall. When participants were ready, they started the sprint from

a line one meter behind the starting gate with a standing start position.

Participants were asked to run at maximum speed and performed the

test twice with a 3 minute rest between trials. Test results were

recorded in seconds and milliseconds, and the best values were used

as 30 m sprint test time (27).

2.9 Ball-kicking speed test

e ball-kicking speed was determined 11 meters (penalty mark)

from the goal using a radar gun device (Bushnell Velocity Speed Gun,

Overland Park, Kansas, UnitedStates), which can measure speed in

the range of 16–177 km/h with a sensitivity of ±2 km/h. Initially, the

dominant legs of the participants were recorded (subjectively

determined); thus, maximal speed was ensured. en, participants

shot the ball with the instep kick technique. Kicks were performed

with a ball (size 5 soccer ball for 12 years and older) in compliance

with FIFA standards. e researcher (the same person aimed the radar

gun in all trials for test reliability) measured the ball-kicking speed

behind the goal, directly opposite the penalty spot where the soccer

player kicked the ball. Participants were told to kick for accuracy

(hitting the target) while attaining the maximum ball-kicking speed.

Each player was given two trials to get the best score, and the results

were recorded in km/h (31).

2.10 Supplementation protocol

Participants consumed 6 mg/kg of caeine (Nature’s Supreme,

Istanbul, Turkey) or placebo supplements (wheat bran) in capsules

(same color and form, made up of gelatin hard form) 60 min before

the testing protocol (6). A researcher, who had no further involvement

in this research, prepared the caeine dose using electronic laboratory

scales with one milligram of sensitivity at room temperature.

2.11 Hydration analysis

e portable hydration test device (MX3 Diagnostics LAB Pro,

Melbourne, Australia) was used to assess participants’ body hydration

FIGURE1

Schematic diagram of the experimental design.

Mor et al. 10.3389/fnut.2024.1359999

Frontiers in Nutrition 05 frontiersin.org

levels (32). e device was convenient and easy to use for outdoor

measurements and analyzed the hydration level from the saliva taken

directly from the tongue with a hydration test strip. e saliva sample

was taken from the tongue under the sterilization rules. e saliva

sample was collected and analyzed with a hydration test strip attached

to the device without waiting or undergoing any other procedure. e

values and their assigned hydration status follow as ≤65 = Hydrated,

65–100 = Mildly Dehydrated, 101–150 = Moderately Dehydrated,

>150 = Severely Dehydrated.

2.12 Statistical analysis

Data were checked for normality by using the Shapiro–Wilk test.

Comparison between groups was analyzed with the paired sample

t-test to test for dierences between the caeine and placebo

supplement in the vertical jump, sprint, reaction time, balance, agility,

and ball-kicking speed performances. Cohen’s d was utilized in the

calculation of eect size (large d > 0.8, moderate d = 0.8 to 0.5, small

d = 0.5 to 0.2, and trivial d < 0.2) (33). Statistical signicance was

accepted as p < 0.05, and all data were analyzed using SPSS 27.0 (IBM

Corp., Armonk, NY) and are presented as mean ± SD.

3 Results

Vertical jump height (p = 0.001, d = 0.83, Table 2) and power

(p = 0.008, d = 0.45, Table2) were signicantly increased aer caeine

compared to placebo supplementation. e Illinois Agility Test

completion time was signicantly faster aer caeine compared to

placebo supplementation (p = 0.008, d = 1.20, Table2). ere was no

dierence in the 30 m sprint (caeine: 4.39 ± 0.14 vs. placebo:

4.42 ± 0.20 s, p = 0.426, d = 0.17), balance (caeine: 2.75 ± 0.48 vs.

placebo: 2.60 ± 0.76 s, p = 0.624, d = 0.23) and ball-kicking speed values

(caeine: 93.87 ± 6.87 vs. placebo: 91.37 ± 5.13 km/h, p = 0.193,

d = 0.41) between supplementations (Table2). RPE values were also

similar between caeine (2.89 ± 0.33 AU) and placebo (2.56 ± 0.72 AU)

supplementations (p = 0.195, d = 0.58) (Figure2).

Regarding reaction time performance parameters, there was a

signicantly faster elapsed time (p = 0.001, d = 1.27, Table3), average

reaction time (p = 0.001, d = 1.37, Table3), and the slowest reaction

time (p = 0.016, d = 1.52, Table3) aer caeine compared to placebo

supplementation. However, there was no signicant dierence in

the fastest reaction time (caeine: 0.71 ± 0.08 vs. placebo:

0.74 ± 0.08 s, p = 0.238, d = 0.37) and in the last reaction time

(caeine: 0.95 ± 0.27 vs. placebo: 1.13 ± 0.30 s, p = 0.097, d = 0.63)

between supplementations.

Fluid balance was within normal ranges in both groups, and there

was no major eect on hydration levels between pre-and post-exercise

conditions (placebo: 53.22 ± 16.57 mOsm/L vs. placebo:

54.78 ± 16.64 mOsm/L, p = 0.863, d = 0.09) (caeine: 54.44 ± 20.92 mOsm/L

vs. caeine: 57.33 ± 20.79 mOsm/L, p = 0.715, d = 0.13) (Table4).

4 Discussion

e aim of this study was to determine the eect of moderate-

dose caeine supplementation on vertical jump, sprint, reaction time,

balance, agility, and ball-kicking speed in the same eld test battery in

moderately trained male soccer players. To the best of our knowledge,

the current study is the rst to analyze the eect of acute caeine

intake on ball-kicking speed, balance, sprint, agility, vertical jump,

reaction time, and hydration status in the same test setting in male

soccer players. e main nding was that the ingestion of 6 mg/kg of

caeine signicantly enhanced vertical jump, agility, and reaction

performance. However, no signicant dierences between placebo

and caeine were detected in hydration status, ball-kicking speed,

balance, sprint performance, and rating of perceived exertion values.

ese ndings are partly in line with our experimental hypothesis and

support a moderate dose of caeine supplementation as an ergogenic

aid to enhance anaerobic performance, including vertical jump height,

COD speed, and reaction time for male soccer players.

e current study ascertained that 6 mg/kg of caeine increased

vertical jump and change of direction performance, which is parallel to

previous studies (9, 34, 35) and is in contrast to the other in which the

participants were recreationally active young males who were not

habituated to caeine (36). is lack of signicant dierence in the

abovementioned study may have resulted from the participants’ status as

non-responders to caeine. However, a performance improvement was

seen with the caeine for 47% of the participants during the 20-yard

shuttle. Our ndings signicantly extend the observations of those

previous studies and support the ergogenic potential of caeine

supplementation on vertical jump and change of direction performance.

e positive eect on jumping performance might berelated to increased

motor unit recruitment (37) and muscle activation (38). Accordingly,

caeine improves performance through two primary mechanisms:

antagonism of adenosine receptors (A1, A2A) in the central nervous

system, which leads to increases in neurotransmitter release and

potentiation of Na

pump activity in skeletal muscle, which may

lead to an increase in excitation-contraction coupling (6). is

mechanism of caeine may also have enhanced jumping performance.

In the study of futsal players, López-Samanes etal. (18) examined the

eects of acute caeine on physical performance. In light of the data

obtained, the researchers suggested that acute caeine supplementation

signicantly improved vertical jump performance. Nevertheless,

although the researchers also found an increase in ball velocity, they did

not nd any signicance. Ranchordas etal. (39) examined the eects of

caeinated gum on vertical jump, sprint, and recovery levels in soccer

players, and they noted that 200 mg of caeine slightly improved physical

performance tests such as jumping and recovery. In line with these

results, acute caeine ingestion of 5 mg/kg 60 min before exercise was

found to increase jump height in professional soccer players (12).

Notably, the researchers found that a caeine dose of 3 mg/kg improved

performance in the majority of the post-exercise tests (10). ese results,

along with our ndings, support the ergogenic eect of acute caeine

supplementation on jumping performance as it was well established in

previous studies (40–42). is eect of caeine on jumping performance

could be attributed to the improvements in force production aer

caeine ingestion (6). Evidently, caeine ingestion has been well-

documented to enhance peak power and mean power and reduce the

time needed to reach peak power in the Wingate test (43). Also, it has

been reported that caeine decreases contraction time and maximal

displacement values, which indicates an increase in muscle contraction

performance. More importantly, this study was carried out using

tensiomyography, in which an electrical pulse induces muscle

contraction independent of the CNS. So, it could be considered

Mor et al. 10.3389/fnut.2024.1359999

Frontiers in Nutrition 06 frontiersin.org

compelling evidence of caeine’s direct eect on neuromuscular

stimulation (44). e improvement in COD time in the present study

might beattributed to the “blocking adenosine” mechanism, which

increases neurotransmission (45) and motor unit recruitment (46). is

change can be elucidated by the fact that caeine intake increases

Ca + and thus improves mobility by facilitating muscle contraction and

nerve conduction (47). Similarly, in their study with rugby players,

Ranchordas etal. (48) investigated the eects of caeinated gum on CMJ,

Illinois Agility, 6 × 30 m repeated sprint, and Yo-Yo IR2 test performances.

Research results indicated that caeinated gum augmented performance

in the Yo-Yo IR2 and the CMJ tests and decreased the fatigue index

during repeated sprints. Furthermore, Karayigit etal. (49) reported that

low (3 mg/kg) and moderate (6 mg/kg) doses of caeinated coee

improved repeated sprint performance with increasing epinephrine

norepinephrine concentrations in female team sport athletes. In contrast,

it was determined that 6 mg/kg acute caeine consumption did not aect

agility and anaerobic power (50). Lastly, it has already been known that

chronic exposure to caeine may result in physiological modications

that lead to tolerance and reduce the ergogenic eects of acute caeine

on high-intensity exercise (16). Because the habitual caeine intake level

of the participants in the current study was not high (188 ± 83 mg/day),

the tolerance phenomenon did not appear in this research.

Considering the present study’s balance and reaction time

performance ndings, it was statistically determined that caeine

intake decreased the reaction time, thus increasing the reaction

performance. Caeine has an adenosine-like molecular structure and

binds to the adenosine receptor (AA2) in the brain, increasing the

concentration of neurotransmitters (51). Also, caeine is a central

nervous system stimulant due to its capacity to block adenosine-

specic receptors, which increases the release of several

neurotransmitters, including norepinephrine, dopamine,

acetylcholine, and serotonin (52). Accordingly, the signicant

dierence in reaction time may have concluded from motivators

through hormonal changes. In two similar studies, Impey etal. (53,

54) reported that caeine consumed 60 min prior to a performance in

soccer goalkeepers positively aected reaction time. Similarly, it was

shown that both 3 and 6 mg/kg of acute caeine intake enhanced

reaction time in female team sport athletes (55). ese decreases in

reaction time could beassociated with the mechanism of caeine as

an adenosine receptor antagonist (6), which may enhance neuronal

excitability that leads to improvements in sport-specic reaction times

(56). Moreover, in our study, increase in reaction performance may

beresponsible for the decrease in change of direction time via the

same mechanism that also makes motor unit recruitment easier.

Hence, caeine may play a signicant role in individual and team

sports where concentration and reaction times inuence match/

training performance. To the contrary, Bottoms etal. (56) showed that

3 mg/kg caeine intake had no eect on reaction time in athletes while

Balko etal. (57) suggested that a larger amount of caeine may lead to

a decrease in visual and auditory reaction times, in turn, increasing

reaction time performance. Interestingly, some results are inconsistent

with our ndings, most likely because the present study enrolled

dierent levels of soccer players than the studies above. e

participants in our study were amateur soccer players with various

training modalities and experiences. erefore, these inconsistencies

in ndings may beprimarily associated with the fact that they are not

familiar with the high demand standardized exercises like professional

soccer players and thus have a dierent level of responsiveness to tests.

Furthermore, according to previous studies, acute caeine

consumption may improve (58) or diminish (59) the standing balance

ability of young adults. is discrepancy may bepartially attributable

to the variance in caeine dosage provided, which ranges from 160 to

400 mg. Only Kara etal. (60) gave a caeine dosage according to body

mass (6 mg/kg); this was the only research that found positive results.

Given the paucity of published research evaluating the inuence of

caeine on balance, it is dicult to contextualize these results in light

of prior research. In addition, methodological inconsistencies and

changes in the balancing tasks and outcome measures used may

possibly account for the inconsistent results. ere is a need for a more

thorough and extensive evaluation of the eects of caeine on balance

skills, based on the small amount of data and inconsistent results.

e ndings of our study indicated that 6 mg/kg caeine

consumption did not induce dehydration, and the athletes’ body

hydration levels were normal. Reviews have shown that this is a

common result, suggesting that no caeine-induced dehydration or

other harmful changes occur in athletes during exercise that negatively

aects physical performance (61, 62). Furthermore, Del Coso etal. (63)

reported that acute caeine consumption of 6 mg/kg increased urine

ow and sweat electrolyte excretion; however, these eects were not

enough to aect dehydration or blood electrolyte levels when exercising

for 120 min in a warm environment. ese results, consistent with our

study’s ndings, refute the widespread belief that caeine consumption

is accompanied by dehydration and an increased sweat rate in the body.

Physiologically, the eects of arginine vasopressin on water retention

and the eects of aldosterone on sodium balance seem to besucient

to tackle the eects of a mild diuretic consumed in a moderate dose

(61). Considering RPE values, caeine is supposed to elicit ergogenic

eects on the CNS via the antagonism of adenosine receptors, leading

TABLE2 Changes in mean values of caeine and placebo groups.

Variables Placebo Caeine 95% CI d p

Х ± SD Х ± SD LB UB

VJ (cm)

56.33±6.87 61.44±5.24

−7.30 −2.92

0.83

0.001*

VJ (watt)

1183.45±105.15 1241.29±146.74

−95.8 −19.87

0.45

0.008*

Balance (s)

2.60±0.76 2.75±0.48

−0.05

0.10 0.23 0.624

30 m sprint (s)

4.42±0.20 4.39±0.14

−0.87

0.55 0.17 0.426

COD (s)

16.43±0.42 15.97±0.34 0.16 0.76 1.20

0.008*

Ball-kicking speed (km/h)

91.37±5.13 93.87±6.87

−6.57

1.56 0.41 0.193

*(p < 0,05); X, mean; SD, standard deviation; d, Cohen’s d eect size; VJ, vertical jump; COD, change of direction (Illionis agility test); 95% CI, condence interval; LB, lower bound; UB, upper

bound.

Mor et al. 10.3389/fnut.2024.1359999

Frontiers in Nutrition 07 frontiersin.org

to pain suppression, which may attenuate the pain and decrease the

RPE (6). Additionally, along with its particular chemical structure,

caeine easily crosses the blood–brain barrier, acts on adenosine

receptors (A1, A2), and blocks the receptors that cause pain in the body.

is is another factor expected to beeective on RPE values in the

present study (64). Intriguingly, wedid not observe a signicant impact

of caeine on RPE levels, indicating that other variables, such as greater

motor neuron activation and a reduced decline in voluntary activation

throughout the exercise, may explain caeine’s ergogenic benets.

e following are some of the limitations of our study. e

blinding eciency was not evaluated by asking individuals to identify

which supplement (caeine vs. placebo) they had ingested. Unknown

is whether “caeine expectancy” may have inuenced the outcomes of

the present investigation. Although participants were advised to repeat

their 24 h meal before each test, macronutrient consumption was not

evaluated. In addition, wedid not collect samples for determining

neurotransmitter concentrations at the baseline and post-exercise, and

wedid not assess the electromyographic activation of active muscles

during performance tests, which would have oered greater insight

into the specic processes by which caeine boosted reaction time,

vertical jump, and change of direction performance but not sprint,

balance, and ball-kicking performance.

FIGURE2

Rating of perceived exertion (RPE) (A.U).

TABLE3 Participants’ reaction time performance parameters.

Variables Placebo Caeine 95% CI d p

X ± SD Х ± SD LB UB

Elapsed time (s) 34.79±1.71 31.92±2.69 1.50 4.24 1.27

0.001*

Average reaction time (s) 1.16±0.05 1.06±0.09 0.06 0.14 1.37

0.001*

Fastest reaction time (s) 0.74±0.08 0.71±0.08

−0.04

0.41 0.37 0.238

Slowest reaction time (s) 1.96±0.31 1.58±0.17

−0.03

0.10 1.52

0.016*

Last reaction time (s) 1.13±0.30 0.95±0.27 0.10 0.65 0.63 0.097

*(p < 0.05); X, mean; SD, standard deviation; d, Cohen’s d eect size; 95% CI, condence interval; LB, lower bound; UB, upper bound.

TABLE4 Hydration levels in both groups between pre- and post-exercise conditions.

Variables Pre-exercise Post-exercise 95% CI d p

Х ± SD (mOsm/l) Х ± SD (mOsm/l) LB UB

Placebo 53.22±16.57 54.78±16.64

−1.96 −1.15

0.09 0.863

Caeine 54.44±20.92 57.33±20.79

−3.60 −2.18

0.13 0.715

*(p < 0.05); X, mean; SD, standard deviation; d, Cohen’s d eect size; 95% CI, condence interval; LB, lower bound; UB, upper bound.

Mor et al. 10.3389/fnut.2024.1359999

Frontiers in Nutrition 08 frontiersin.org

5 Conclusion

e present study found that a 6 mg/kg caeine intake in soccer

players improved physical performance, such as vertical jump height,

change of direction speed, and reaction time. Although

non-signicant, caeine intake also improved sprint (0.67%) and ball

kicking (2.7%) performance percentages. Moreover, it was revealed

that caeine did not impair body hydration levels, so body uid

balance was within normal clinical ranges. Accordingly, further

studies conducted with larger sample sizes using a 24 h urine collection

method could provide more evidence for the relationship between

caeine consumption and hydration. On the other hand, in our study,

caeine ingestion did not provide any change in balance performance

and rating of perceived exertion. Based on these results, it is

recommended that coaches and athletes incorporate caeine into their

nutritional strategies as it can improve performance. In future studies,

the eects of dierent doses of caeine on various components of

performance, including small-sided games and ball-kicking speed in

soccer and other disciplines, could beinvestigated to provide new

insights. Ultimately, to verify these mechanisms, further studies

should be conducted to investigate neuromuscular responses to

caeine supplementation during anaerobic tasks in soccer players.

Data availability statement

e raw data supporting the conclusions of this article will

bemade available by the authors, without undue reservation.

Ethics statement

e studies involving humans were approved by Human Research

Ethics Committee at Sinop University (Reference number: E-57452775-

050.01.04-104421). e studies were conducted in accordance with the

local legislation and institutional requirements. e participants

provided their written informed consent to participate in this study.

Author contributions

AM: Conceptualization, Data curation, Formal analysis,

Methodology, Project administration, Supervision, Writing – original

dra, Writing – review & editing. KA: Data curation, Formal analysis,

Writing – review & editing. DA: Funding acquisition, Methodology,

Project administration, Soware, Supervision, Writing – review &

editing. HM: Methodology, Writing – original dra, Writing – review

& editing. MA: Project administration, Supervision, Writing – original

dra, Writing – review & editing. MM: Data curation, Funding

acquisition, Soware, Visualization, Writing – review & editing. FK:

Data curation, Writing – review & editing. FW: Funding acquisition,

Soware, Visualization, Writing – review & editing. AY: Formal

analysis, Supervision, Writing – review & editing. CA: Data curation,

Funding acquisition, Soware, Supervision, Visualization, Writing –

review & editing.

Funding

e author(s) declare that no nancial support was received for the

research, authorship, and/or publication of this article.

Acknowledgments

Great thanks go to all collaborators and volunteers for their

availability and contribution to this study. CA and DA are thankful for

the support and assistance provided by the Vasile Alecsandri

University of Bacau, Romania.

Conﬂict of interest

e authors declare that the research was conducted in the

absence of any commercial or nancial relationships that could

beconstrued as a potential conict of interest.

Publisher’s note

All claims expressed in this article are solely those of the authors

and do not necessarily represent those of their aliated

organizations, or those of the publisher, the editors and the

reviewers. Any product that may beevaluated in this article, or claim

that may bemade by its manufacturer, is not guaranteed or endorsed

by the publisher.

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