Acute Effects of Combined and Isolated Caffeine and Theanine Supplementation on Physical and Cognitive Performance in Competitive Athletes: A Randomized, Double-Blind, Placebo-Controlled Crossover Study
Phase: NA
Conditions: Cognitive Performance, Physical Performance Enhancement, Caffeine and L-theanine Interaction
Sponsor: Ulaş Can YILDIRIM

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STUDY PROTOCOL – COVER PAGE 
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Official Title: 
Acute Effects of Combined and Isolated Caffeine and Theanine Supplementation on Physical and Cognitive 
Performance in Competitive Athletes: A Randomized, Double-Blind, Placebo-Controlled Crossover Study 
 
Sponsor-Investigator: 
Assoc. Prof. Ulaş Can Yildirim  
Faculty of Sport Sciences, Sinop University, Türkiye 
ucyildirim@sinop.edu.tr 
 
Ethics Committee Approval: 
Sinop University Human Research’s Ethics Committee 
Approval Number: 2025/337 
Approval Date: 22 May 2025 
 
Document Type: 
Study Protocol 
 
Version and Date: 
Version 1.0 – October 21, 2025 
 
Funding Source: 
Self-supported academic research 
 
 
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Method 
Study Design  
This study employed a randomized, double-blind, placebo-controlled, crossover design to examine the effects of caffeine 
(CAF), theanine (THE), their combination (COM), and a placebo (CON) on physical and cognitive performance in young, 
healthy, competitive athletes. Each participant completed all four experimental conditions in a randomized order, with a 
washout period of at least 72 hours between sessions to minimize potential carryover effects. The randomization sequence was 
generated by an independent researcher using a computer-based randomization tool (Urbaniak & Plous, 2013). To ensure 
familiarity and minimize learning effects, all participants completed a familiarization session one week prior to the experimental 
trials. Physical and cognitive performance were assessed across multiple primary outcomes, including maximal strength, 
intermittent aerobic endurance, and eye-hand coordination. All procedures were conducted in accordance with the of Helsinki. 
The study protocol was approved by the Sinop University Human Research’s Ethics Committee (Approval No: 2025/337), and 
all participants provided written informed consent prior to participation. 
Participants 
12 males and 8 females athletes (19.9 ± 2.4 years; 167.3 ± 7.6 cm height; 63.5 ± 10.2 kg body mass; 27.5 ± 6.7 kg lean body 
mass; 22.6 ± 8.2% body fat; 22.6 ± 2.3 kg·m⁻² BMI) from both team and individual sports were recruited for the study (Table 
1). During the first laboratory visit, anthropometric and body composition assessments were performed to characterize the 
participant sample. Standing height and body mass were measured to the nearest 0.1 cm and 0.1 kg using a calibrated 
stadiometer and digital scale (Seca, Hamburg, Germany). Body composition was determined via multi-frequency bioelectrical 
impedance analysis (InBody 770; InBody Co., Seoul, South Korea). All measurements were conducted in the morning, with 
participants wearing light clothing and having abstained from exercise, food, and caffeine for at least 3 hours prior to testing. 
Inclusion criteria were: (i) active participation in individual or team sports at a competitive level, (ii) age between [insert range, 
e.g., 18-30] years, and (iii) absence of any known medical or metabolic disorder. Exclusion criteria included: (i) history of 
cardiovascular, neurological, or musculoskeletal disorders within the past 12 months, (ii) known intolerance or hypersensitivity 
to caffeine or theanine, (iii) allergy to mannitol or other excipients used in capsule formulation, (iv) current use of prescription 
medications, and (v) physician-directed restriction of caffeine or theanine consumption. 
 
Figure 1. Flowchart 
 
Responses 
(n = 24) 
Familiarized Consented 
(n = 20) 
Randomized 
(n = 20) 
CAF 
Condition 
N = 5 
N = 5 
N = 5 
N = 5 
Crossover 
Round 
1 
Failed to meet inclusion 
criteria (n = 4) 
Analyzed 
(n = 20) 
THE 
Condition 
COM 
Condition 
CON 
Condition 
N = 5 
N = 5 
N = 5 
N = 5 
Round 
2 
N = 5 
N = 5 
N = 5 
N = 5 
Round 
3 
N = 5 
N = 5 
N = 5 
N = 5 
Round 
4 

The required sample size was estimated using an a priori power analysis (G*Power 3.1.9.7; Düsseldorf, Germany) for a 
repeated-measures ANOVA with four conditions (CAF, THE, CAF + THE, CON), assuming a moderate effect size (f = 0.25), 
α = 0.05, and statistical power (1 - β) = 0.80. The analysis indicated a minimum required sample of 16 participants. To account 
for potential attrition or missing data, 20 athletes were recruited; all completed the study protocol. 
Supplementation Protocol 
Capsules were ingested 60 minutes prior to testing to allow sufficient time for peak plasma caffeine concentrations (Pallares et 
al., 2013). The supplementation conditions were structured as follows: caffeine (CAF, 3 mg/kg⁻¹ anhydrous caffeine), theanine 
(THE, 200 mg L-theanine), combined caffeine and theanine (COM, 3 mg/kg⁻¹ caffeine + 200 mg theanine), and placebo (CON, 
mannitol). Caffeine capsules contained anhydrous caffeine powder (Oxford brand pure caffeine powder; ISO 14001 certified; 
98.5% purity; The Oxford Vitality Health Company Ltd., London, UK), and theanine capsules contained pure L-theanine 
powder Ocean (L-Theanine, 200 mg; Orzax Pharmaceuticals, Istanbul, Turkey; active ingredient manufactured in Germany). 
Placebo capsules contained mannitol, a non-nutritive sugar alcohol with no known ergogenic or ergolytic properties (Flueck et 
al., 2016). All supplements were administered in visually identical gelatin capsules to ensure blinding across conditions.  Each 
participant ingested the same number of capsules (five in total) in all conditions to maintain consistency in capsule count and 
total mass. Neither participants nor researchers could distinguish between conditions due to the capsules’ identical appearance, 
texture, and color.  
Participants were instructed to refrain from alcohol consumption and strenuous exercise for 24 hours before each trial and to 
abstain from any dietary supplements throughout the study period. They maintained 24-hour dietary records for the day 
preceding each test and a weekly log of their habitual caffeine intake. To standardize pre-trial nutritional status, participants 
replicated their recorded dietary intake and hydration patterns before each visit. To preserve ecological validity and reduce 
potential caffeine withdrawal effects, participants were permitted and encouraged to maintain their usual daily caffeine 
consumption during the study period, as recommended by Tallis et al. (2021) and Pickering & Kiely (2019). 
Data Collection Tools 
All sessions were conducted under standardized computer laboratory and indoor sports hall conditions. The eye-hand 
coordination assessment was conducted in a quiet, temperature-controlled laboratory using a desktop computer system. 
Ambient conditions were maintained at approximately 22 ± 1 °C and 50 ± 5% relative humidity. All other performance tests 
(strength and aerobic endurance) were carried out in an indoor sports hall under comparable conditions (temperature 21-23 °C, 
relative humidity 45–55%). Environmental parameters were monitored at the start of each session using a calibrated digital 
room thermo-hygrometer. Lighting, background noise, and testing times were standardized across all trials to ensure intra-
individual consistency. 
Testing sessions were held in the morning at 09:00 a.m. to minimize the potential effects of circadian variation and diurnal 
performance fluctuations. The sequence of tests was standardized as follows: eye-hand coordination, maximal strength, and 
aerobic endurance. Adequate rest intervals were provided between tests to prevent fatigue and carry-over effects. The following 
measurements were collected during each experimental condition: 
Strength Tests 
Maximal isometric strength was assessed using a Lafayette digital dynamometer (Takei Scientific Instruments Co. Ltd, Tokyo, 
Japan) to measure handgrip, back, and leg strength. The device was checked for calibration prior to each testing session 
according to the manufacturer’s recommendations. 
Participants performed three maximal voluntary contractions for each test, with at least 60 seconds of rest between attempts to 
minimize fatigue. Handgrip strength was assessed with participants seated, shoulders adducted, elbows flexed at 90°, and the 
dynamometer handle adjusted to individual hand size. Back strength was measured in a standing position with the participant 
pulling upward on a handle attached to the dynamometer’s base platform while maintaining a neutral spine. Leg strength was 
evaluated in a similar posture, with participants exerting maximal upward force through a chain-linked platform while keeping 
their knees slightly flexed. 
The mean of the three trials for each test was recorded as the participant’s strength score, expressed in kilogram-force (kgf) as 
displayed by the device. All strength assessments were performed in accordance with the standardized procedures for isometric 
testing recommended by the American College of Sports Medicine (ACSM, 2021). American College of Sports Medicine. 
(2021). ACSM's Guidelines for Exercise Testing and Prescription (11th ed.). Wolters Kluwer. 
Aerobic Endurance 
Aerobic endurance was assessed using the Yo-Yo Intermittent Endurance Test Level 1 (Yo-Yo IET-1), which evaluates 
participants’ ability to perform repeated bouts of exercise with brief recovery periods. The protocol followed the standardized 
procedures described by Bangsbo et al. (1996). Testing was performed on a flat, non-slip indoor surface with two parallel lines 
marked 20 m apart and a recovery line placed 5 m behind the starting line. Prior to testing, participants completed a standardized 
5-minute dynamic warm-up consisting of jogging and mobility drills. Bangsbo, J. (1996).  

Participants ran back and forth between the 20-m lines in time with audio cues from the official Yo-Yo IET Level 1 recording, 
with running speed progressively increasing throughout the test. After each 2 × 20 m shuttle, a 5-second active recovery period 
was provided, during which participants jogged to and from the recovery line. The test was terminated when a participant failed 
to reach the 20-m line on two consecutive occasions, and the total distance covered (m) was recorded to the nearest meter as 
the performance outcome. 
Eye-Hand Coordination 
Eye-Hand Coordination Test (Multidirectional and Unpredictable Direction) was used to evaluate participants’ ability to 
perform accurate, continuous manual motor actions based on dynamic visual processing. The test was administered in a quiet, 
temperature-controlled laboratory (22 ± 1 °C; relative humidity 50 ± 5%) using CogniFit’s proprietary digital platform 
(CogniFit Inc., New York, USA) on a desktop computer. 
They were instructed to remain seated comfortably, maintain a neutral wrist position, and use their dominant hand for all trials. 
During testing, participants tracked a small moving circle (“ball”) that followed an unpredictable multidirectional trajectory. 
The task required maintaining a cursor (crosshair) within the moving circle as precisely as possible. Visual feedback was 
provided continuously: green when on-target, orange when near the boundary, and red when the cursor left the target area. Each 
assessment included two initial learning segments (7 s slow + 7 s fast) followed by twelve testing segments alternating between 
slow (≈5 cm/s) and fast (≈10 cm/s) movement speeds, each lasting either 2333 ms (short) or 7000 ms (long), for a total duration 
of approximately 5-7 minutes including instructions. Prior to the main test, participants completed a brief familiarization session 
that included standardized on-screen practice trials lasting approximately 1 minute to minimize learning effects. Optional 10-
15 second rest intervals were allowed between trials to avoid fatigue. 
The following performance metrics were recorded automatically by the CogniFit software: 
Accuracy (%): Percentage of time the cursor remained inside the circle (0–100; higher = better). 
Accuracy - Fast Speed (%): Accuracy during fast segments (≥ 10 cm/s). 
Accuracy - Slow Speed (%): Accuracy during slow segments (≤ 5 cm/s). 
Accuracy - Long Segments (%): Accuracy during 7000 ms segments (sustained tracking). 
Accuracy - Short Segments (%): Accuracy during 2333 ms segments (brief tracking). 
Distance from Circle Center (pixels): Mean pixel distance between cursor and target center (0–1200; lower = better 
precision) 
Performance scores were automatically normalized against CogniFit’s age- and sex-matched normative database (percentile 0–
100), following the developer’s validity index ranges and completion-time criteria (54–58 s valid window). All assessments 
adhered to the manufacturer’s standardized task protocol and validity thresholds (CogniFit Inc., 2023). 
Statistical Analysis   
The normality of the data set was initially evaluated using the Shapiro-Wilk test. Subsequently, the assumption of sphericity 
was examined through Mauchly’s test, with the Greenhouse-Geisser correction applied in cases where sphericity was violated. 
Differences in strength, aerobic endurance, and eye–hand coordination outcomes across the four supplementation conditions 
(CAF, THE, COM, and CON) were analysed using a one-way repeated-measures analysis of variance (ANOVA). When 
significant main effects were observed, Bonferroni-adjusted pairwise comparisons were conducted to identify specific between-
condition differences. The magnitude of effects was quantified using partial eta squared (ηp²), interpreted as small (0.10–0.24), 
moderate (0.25–0.39), or large (≥0.40). For pairwise comparisons, Cohen’s d for repeated measures and corresponding 95% 
confidence intervals (CI95%) were reported and interpreted as trivial (<0.20), small (0.20–0.49), moderate (0.50–0.79), or large 
(≥0.80) (Cohen, 1992). All statistical analyses were performed using IBM SPSS Statistics version 30.0 (IBM Corp., Armonk, 
NY, USA) and statistical significance was accepted at α = 0.05 (two-tailed). Graphical visualizations were generated with 
GraphPad Prism version 10.0 (GraphPad Software, San Diego, CA, USA).