The study aims at assessing the relation between caffeine (CAF) and paraxanthine (PAX) kinetics after acute ingestion of caffeine (3 mg/kg body mass) in the form or mono- or multi-ingredient preparations and CrossFit®-specific performance or selected indicators of psychophysiological state. Blood indicators of muscle damage/recovery, acid-base balance and electrolytes levels will be evaluated. The impact of genetic variations in the polymorphisms rs5751876 in ADORA2A gene and rs762551 in CYP1A2 on CAF/PAX kinetics will be considered.

The main aim of the project is to assess blood and saliva kinetics of CAF and PAX after acute ingestion of caffeine (3 mg/kg body mass) in the form of mono-ingredient preparation (CAFMONO) or two different multi-ingredient pre-workout supplements (CAFMIPS_1 CAFMIPS_2) compared to Placebo (PLA). The impact of CAF/PAX kinetics on CrossFit®-specific performance (as measured via the Fight Gone Bad test <FGB>) and selected psychophysiological indicators such as reaction time, postural stability, the level of focus, alertness, fatigue and feeling of 'energy', as well as the rate of perceived exertion (RPE) will be evaluated. Additionally, blood indicators of muscle damage/regeneration (haematological evaluation; activity of: creatine kinase <CK>, lactate dehydrogenase <LDH>, aspartate aminotransferase <AspAT> and alanine aminotransferase <AlAT>; levels of creatinine <Cre> and mioglobin), acid-base balance (pH, levels of: bicarbonate, , lactate <La>, anion gap; base excess, H+) and concentrations of electrolytes (Ca2+, Na+, K+, Cl-) will be evaluated. Saliva and blood parameters will be assessed before the test preparations ingestion (-10minINT), after ingestion (+15, +30, +45, +60 minINT) and after FGB (+3, +10 minFGB). The indicators of psychophysiological state will be evaluated at -10minINT, +30, +60 minINT, +10minFGB.

The study is a double-blind, randomized, placebo-controlled, cross-over trial. The study consists of preliminary meeting with research team (familiarization with study protocol) and five study visits (T0 - T4), of which T0 is a BLANK visit with no supplementation and supplementation visits (T1 - T4). The testing days will be separated with 7-days wash-out. Investigators aimed to recruit 25 male CrossFit®-trained participants. Random allocation of participants to supplementation order (PLA, CAFMONO, CAFMIPS_1 CAFMIPS_2) will be done using a random sequence generator, with participants being equalised by fat-free mass and number of repetitions in FGB test (established at BLANK visit).

At the preliminary (familiarization) visit the written informed consent for participation in the study protocol will obtained from all participants. After that, anthropometric measurements and familiarization with exercise test procedures (Fight Gone Bad test) will be performed. The biological samples for evaluation of polymorphisms rs5751876 in ADORA2A gene and rs762551 in CYP1A2 will be obtained. At the BLANK visit (T0) no supplementation will be provided. However, the remaining study procedures (anthropometric measurements, blood and saliva collection, FGB and psychophysiological evaluation) will be performed. After the completion of T0 visit, the participants will be randomly assigned to the order of supplementation.

Each of T1-T4 visits will start with body mass and body composition evaluation (electric bioimpedance method). Then the baseline (resting) blood and saliva samples, as well as psychophysiological evaluation will be done (-10minINT). Ten minutes after the baseline evaluation, each participant will ingest the individualized dose of PLA or CAFMONO or CAFMIPS_1/CAFMIPS_2. The doses of the test preparations will be adjusted so that they will provide 3 mg/kg BM of CAF (apart from PLA, which will be taste- and colour-matched but caffeine-free preparation). Sixty-minutes after test preparations ingestion, participants will perform the test exercise - FGB. FGB test comprised of three rounds of five exercises: wall ball, sumo deadlift high pulls, box jump, push press and rowing. Participants are obligated to complete as many repetitions as possible in one minute at each station prior to moving to the next station. After completing each of the five stations, participants will have one minute of rest. The total time to complete FGB test will be17 minutes. After the test preparations intake, before and after FGB the blood and saliva samples drawn, and evaluation of indicators of psychophysiological state will be performed according to the schedule mentioned above.

After collection all the data, comparisons between supplementation treatment conditions (BLANK, PLA, CAFMONO, CAFMIPS_1, CAFMIPS_2) will be performed using repeated measurements analysis of variance (RM ANOVA). Additional models considering gene polymorphisms or treatment order will also be analysed. The level of significance adopted will be p <0.05. Statistical analysis will be performed using Statistica v. 13.3, StatSoft Polska Sp. z o.o., 2022. G*Power software (version 3.1.9.4, Universitat Dusseldorf, Germany) was used to calculate sample size required to obtain a power of approximately 80% (α = 0.05) and large effect size partial eta squared 0.14 in RM ANOVA within factors. Analysis indicated that a sample size of 9 is suitable for detecting differences between five measurements. To account for possible drop-out, the sample size at 25 is established.

Inclusion Criteria:

• written informed consent from all participants before the study,
• a current medical clearance to practice sports,
• training experience any sport discipline ≥ 5 years and training experience in HIFT ≥ 2 years,
• performing ≥3 training units per week,
• participating in CrossFit® competitions at least once a year.

Exclusion Criteria:

• current injury,
• any health-related contraindication,
• declared general feeling of being unwell,
• unwilling to follow the study protocol,
• serious disease or metabolic problems,
• smoking and tobacco use,
• presence of infectious disease in the previous 4 weeks of the study.