This randomized, double-blinded, placebo-controlled, within-subjects counter-measured
study design will include 60 males and females that fall between the given age range
(18-35 years).
Participants will complete five visits (~90-120 min each, 2 visits during the first
week,3 visit during week 2-4) in the Laboratory at Faculty of Sports Studies of Masaryk
University. All testing will take place Monday to Friday during morning hours (7-12) to
minimize potential diurnal effect on sports performance. Participants will be instructed
to maintain their regular diet (pre-testing meals will be replicated for all visits) and
sleeping habits, avoid strenuous activity 48 hours before each visit, and abstain from
caffeine one week prior to the first visit and for the duration of the data collection (4
weeks).
Sample size calculations We estimated sample size using G*Power 3.1 (Version 3.1.9.4).
Effect size = 0.25, the trial would be powered to detect small but still physiologically
important effect.
Alpha error = 0.025 Power (1-Beta probability) = 0.8 Number of groups = 6 - number of
stratification required for genotype subgroup analysis number of measurements = 3 Sample
size = based on calculation, 60 participants are required
Recruitment
Recruitment will be carried out at Masaryk University by flyers. Recruitment will also
take place online through sharing on the website and social networks of the Faculty of
Sports Studies of Masaryk University.
Parameters of Assessment
Physiological data will be collected by a Ph.D.-level exercise physiologist and two
doctoral students.
Height will be measured to the nearest 0.1 cm using a wall-mounted stadiometer, and body
weight will be measured to the nearest 0.1 kg using a digital scale with participants
wearing light clothing without shoes.
Body weight value will be measured on each visit and will be used to determine 2 and 4 mg
per kilogram of body mass dosages for caffeine treatments (plus a placebo dose) that will
be ingested in random order (derived by research assistant and blinded to all
investigators) during visits 2, 3 and 4, over the following three weeks.
Body composition will be assessed by dual-energy x-ray absorptiometry (DXA; Horizon A,
Hologic Inc. Marlborough, MA) during the first week of the study.
Exercise Tests
VO2max test
Each participant will initially perform an incremental exercise test. That test will
assess Vo2max and maximal workload (Wmax), which is necessary for subsequent settings.
Respiratory parameters will be measured breath-by-breath using a CORTEX METALYZER 3B.
Wmax will be calculated using the following equation:
Wmax= Wfinal + (t/T) x Winc. Where Wfinal is the power output of the final completed
stage, t is the time achieved in the final uncompleted stage (s), T is the duration of
each stage (s) and Winc is the workload increment.
Time trial
The ergometer will be set to linear mode (workload increases as the pedalling rate
increases). Each participant will perform a warm-up at 1 W/kg for 5 minutes. After the
warm-up participants will be instructed to complete 10 km trial as quickly as possible
(with no temporal, verbal, or physiological feedback). The primary variable measured will
be the time to completion of the 10 km TT. The ergometer will be set to linear mode using
the following formula:
W = L x (rpm)2, where rpm is the pedalling rate and L is a linear factor dependent on the
participant's Wmax. To ensure consistency across participants, the resistance on the
pedals during the TT will be set so that the participants would achieve 60 % of their
power output at 80 rpm using the following equation:
L = (0.60 x Wmax)/6400
A familiarization time trial will occur during the first week of a study.
Rate of perceived exertion
During the time trial protocol, information about the rate of perceived exertion will be
collected. Participants will be shown a Borgs scale of Perceived Exertion to assess the
subject's fatigue, This will be performed twice during the time trial protocol (5th and
9th km)
Genotyping
Buccal mucosa smears will be collected during the first week of the study. Investigators
will examine known genetic variations (CYP1a2 (rs 762551), ADORA2A (rs 5451876)) that are
associated with caffeine metabolism and response, to determine if these genes affect
response to exercise after subjects ingest caffeine.
Caffeine intake questionnaire
Investigators will use a Caffeine intake questionnaire - revised (CCQ-R) to determine
habitual caffeine intake in the week before the study starts and then each week to
discover whether the participants comply with the restriction regarding caffeine, or
whether they have started consuming, for example, more chocolate
Training status
At the beginning of the study, participants with one of the study investigators will
complete Training and Performance Caliber: A Participant Classification Framework, based
on the study by McKay et al., 2021 to determine the training status of each individual.
Statistical analysis
The statistical analysis will be carried out using IBM SPSS software. Descriptive data
will be reported (VO2max/peak, body height, body weight, %body fat, lean body mass,
habitual caffeine intake, training status. A t-test will determine the comparison between
genotypes. Potential differences between 10 km TT will be assessed by an ANOVA with
treatment as a within-participants factor and genotype as a between-participants
factor.if the distribution of the data will not be normal, we will apply non-parametric
equivalent of the test
Experimental procedure
Week 1
There will be three visits to the laboratories of the Faculty of Sports Studies at
Masaryk University (FSpS MU):
i. Participants will undergo body composition measurement (DXA) and a cheek swab for
assessing selected gene variants. Data about height, weight and age will be collected
(Day 1: approximate duration of visit to FSpS MU 60-90 minutes).
ii. Participants will undergo maximal load testing using a cycling ergometer test and
critical power testing using a cycling ergometer test (Day 2: approximate duration of
visit to FSpS MU 90 minutes).
iii. Participants will practice a 10-km time trial on the cycling ergometer (Day 3:
approximate duration of visit to FSpS MU 60 minutes).
Weeks 2, 3, 4
Participants will undergo a 10-km time trial on the cycling ergometer. After arrival
participants will be randomly assigned to one of the three conditions. They will always
ingest same amount of the identical capsules (with same colour, size). After 50 minutes
after the capsule ingestion, participants will start the warm-up and 60 minutes after the
ingestion the TT will start. This procedure will be identical for all experimental week
(2, 3 and 4). Participants may drink water ad libitum throughout the exercise test.