Effects of Caffeine Ingestion on Morning Cognitive and Muscle Strength Measures in Males

Participants Fourteen male adults of biological sex and gender who were classified as "recreationally active". All participants were recruited from the University Sport Science department student population. Inclusion criteria were healthy adults (18-30 years), injury-free, with previous weight/strength training experience (≥ 2 years). The project also opened participation to females, but no females volunteered. Participants habitually retired to bed between 22:00-23:30 h and rose at 06:00-07:30 h and for the study purposes agreed to retire at 22:30 and rise at 06:30 h. None of the participants were receiving any pharmacological treatment (including non-steroidal anti-inflammatory drugs, NSAIDs) throughout the study period. Habitual caffeine consumption was assessed using the caffeine consumption questionnaire (CCQ) and those with >150 mg per day were excluded. Further, all participants expressed no preference to training regarding time-of-day by a weekly self-reported 2-week training diary. Recruiting participants with this specific type of exercise history meant that the known neuromuscular facilitative responses, which are typically associated with acute increases in muscular strength amongst untrained individuals due to neural adaptations and responses were reduced. Exclusion criteria included depressed mood (from the Beck depression inventory), poor sleep quality (a Pittsburgh sleep quality index global score >5, recent shiftwork or travel across multiple time-zones and 'extreme' chronotype (assessed via the Compo-site Morningness Questionnaire) and risk factors and/or symptoms of cardiovascular disease. Through interviews, it was established that the volunteers had minimal knowledge of the effects of time-of-day or time-since-sleep on human performance. Verbal explanation of the experimental procedure was provided to everyone; this included the aims of the study; the possible risks associated with participation and the experimental procedures to be utilized. Any questions were answered. Individuals then provided written, informed consent before participating in the study. The experimental procedures were approved by the Human Ethics Committee at Liverpool John Moores University (ETHICS CODE: 08/SPS/030). The study was conducted in accordance with the ethical standards of the journal and complied with the principles of the Declaration of Helsinki.

Research design All participants were required to visit the physiological laboratory (Liverpool, UK) on seven occasions (dry temperature of 19-21°C, 29-41% humidity, barometric pressure of 962-1010 mmHg and ambient light of 750 lux). Participants completed (i) one maximum repetition (1RM) for bench press and back squat, ii) two familiarization sessions of cognitive and strength performance tests one week prior to the experimental protocol. During familiarizations participants completed a 7-day food diary that was analyzed using Nutritics® analysis software by a SENr registered Sports and Exercise Nutritionist. The remaining sessions consisted of iii) three experimental sessions, all at least 72-h apart as a standardized recovery period. The experimental part comprised three experimental trials a) Control (NoPill), b) Caffeine (300 mg, CAFF) and c) placebo (maltodextrin, PLAC)] between 07:30 and 08:30 h (Figure 1). Participants retired the previous night around 22:30 h, woke up at 06:30 h and arrived at the laboratory at 07:00 h in a fasted state. For the CAFF and PLAC conditions, pills were provided in a plastic bottle with instructions to consume with water at 06:30 h prior to arrival at the laboratory, with confirmation upon arrival. For the NoPill control, nothing was provided. Participants were instructed to avoid training or intense physical activity 48 h prior to any of the visits, other-wise, participants maintained their normal daily routines. In total the three caffeine capsules contained 300 mg of caffeine anhydrous and the remaining space was occupied by malto-dextrin (Applied Nutrition LTD, Knowsley, UK) and placebo capsules were made in the department and contained malto-dextrin (~2.4 g, Sport supplements Ltd t/a BulkTM Colchester, UK). Researchers and participants were blinded to the supplement schedule and both caffeine and placebo were lightly dusted with malto-dextrin to create a similar taste, both had similar weight (0.8 g/capsule) and were 00 size. The order for treatment was revealed at the end of the project by a researcher responsible for anonymization and randomization.

All trials were performed with only one participant at a time, but with a staggered start so one participant came in at 06:45, the next 07:00 h and the last for that morning's session at 07:15 h. This scheduling for participants was kept consistent for all sessions. All participants lived no more than 10 mins away from the University laboratories. Participants were equally allocated, based on their 1RM for bench and back squat into three groups (A, B and C), by listing participants' times from the second familiarization in order from the strongest to the weakest and assigning letters A, B and C in order. The incidence of the learning effect was minimized by assigning the experimental conditions in counterbalanced order. The present study was carried out between the months of November to February (UK Autumn to Winter), with morning sunlight exposure limited to <80 Lux before arriving at the laboratory. The sunrise range for the duration of the study was 07:12 to 07:03 h.

Protocol and measurements: familiarization sessions The participants completed two familiarization sessions of all the strength, cognitive and questionnaire measures, before being considered as ready to participate in the study. A coefficient of variation values less than or equal to 10 % for variables between the first and second measures was used as criteria for familiarization in alignment with the recommendations given by Atkinson and Nevill [26]. Familiarization sessions took place at 12:00 h over a three-week period and finished one week before the study commenced to minimize learning effects. Participants arrived 0.5 hours before the start of the test and rested in a seated position, to minimize the influence of prior muscle activity. Participants completed the familiarization in the following order: i) cognitive battery of tests, ii) isometric maximal voluntary contraction (MVC) with and without percutaneous electrical stimulation, iii) back squat and bench press.

1. Cognitive battery of tests Trail Making Test (TMT; parts A and B): Both parts of the TMT consist of 25 circles distributed over a sheet of A4 paper. In part A the circles are numbered 1-25, and the participant is instructed to draw lines to connect the numbers in ascending order. In part B, the circles include both numbers (1-13) and letters (A-L) and the participant is in-structed to draw lines to connect the circles in ascending pattern but with the added task of alternating between numbers and letters (i.e., 1-A-2-B-3-C etc.). In both parts the participant is instructed to connect to the circles as fast as possible, without lifting the pencil from the paper. If an error is made, this is pointed out immediately and the participant is allowed to correct it. During the test, time to completion is measured, with a higher time indicative of the greater impairment.

Rey Auditory Verbal Learning Test: The RAVLT is a neuropsychological assessment designed to evaluate verbal memory in patients aged 16 years and older. The RAVLT can be used to evaluate the nature and severity of memory dysfunction and to track changes in memory function over time [28]. The test is designed as a list-learning paradigm in which the volunteer hears a list of 15 nouns and is asked to recall as many words from the list as possible. The number correct and the number that were given by the participant but not on the list (intrusions) are noted. This process is re-peated 4 more times. The process is repeated but with a second "interference" list (List B) is presented in the same manner, and the participant is asked to recall as many words as possible from List B and scoring recorded. After the interference trial, the participant is immediately asked to recall the words from List A, which they heard five times previously and the number of correct words and intrusions are recorded. RAVTL total number, number of distractions and retention are recorded and analysed.

Stroop word-colour interference test. The participants were asked to read out their responses to words or colours for 45 s as quickly as possible and to leave no errors uncorrected. This was filmed and the number of errors and total amount completed was recorded and analysed. The first sheet had text (red, blue, yellow, black and green) in black ink (naming word test, W). The second sheet had blocks of colour corresponding to the text on the first sheet (naming colour test, C). With the third sheet, the participants had to read out the word (which was coloured differently to the word, e.g., the word was yellow and the colour red, referred to as the naming colour of word test, CW) and for the fourth sheet, the participants had to read out the colour (which was wrongly named, e.g., the colour was yellow but the word was red, referred to as the naming of word not colour test, WC). In this fourth sheet, the words were printed in the reverse order to the third sheet. The raw data were analysed for number of errors (to give an indication of pacing/speed accuracy in the 45 s) and interference scores (an indicator of the efficiency of the inhibitory function), where C represents the correct answers produced in 45 s in naming colours, and CW corresponds to the correct answers achieved in 45 s in the interference condition. This was also performed for the naming of word not-colour test.

I = [(C - CW) / (C + CW)] × 100 (1)

ii) Isometric maximal voluntary contraction Participants performed isometric MVCs of the quadriceps muscles (3 s duration), both with and without twitch interpolation percutaneous stimulation (Digimeter, DS7, Hertfordshire, UK). During the initial session, participants practiced performing MVCs without twitch interpolation, to become accustomed to the practice of achieving and maintaining voluntary force for the time required. This session was also used to obtain maximal current tolerance and establish the supra-maximal current amplitude for superimposition during an MVC. With the subject at rest, the amperage of a 240 V square-wave pulse (100 μs, 1 Hz) was progressively increased until the point when further increases in intensity caused no further increase in resting twitch force reached.

Twitch interpolation The quadriceps were electrically stimulated using two moistened surface electrodes (Chattanooga, USA, 5 x 12 cm) which were positioned proximally over the vastus lateralis and distally over the vastus medialis. The skin was pre-pared prior to placement of each electrode by shaving and light abrasion of the skin, followed by cleaning with an iso-propyl alcohol swab. A permanent marking pen was used to mark and identify the position of each electrode to minimize electrode placement variability from session to session. Two impulses were delivered before and after the contractions; the other two impulses were administered during the contraction period and tested the peak value of the MVC. The peak forces of the pre- and post-contraction twitches were then averaged, allowing comparison of resting twitch amplitudes in both an unpotentiated and potentiated condition respectively. The amplitude of supra-maximal superimposed current was identified for each subject in familiarization sessions and corresponded to 10% above the level required to evoke a resting muscle twitch of maximal amplitude.

Data was acquired for nine seconds and analysed with a commercially designed software program (AcqKnowledge III, Biopac Systems, Massachusetts). The calculation of voluntary activation was conducted according to an interpolated twitch ratio whereby the size of the interpolated twitch is ex-pressed as a ratio of the amplitude elicited by the same stimulus delivered to a relaxed potentiated muscle. The average force during the 100 m.s-1 period before the application of each stimulus during the contraction was recorded and subsequently the maximal force recorded during the 100 m.s-1 period after each stimulus had been applied. The 100 m.s-1 mean pre-stimulus force (taken as MVC force) and the resulting maximal post-stimulus force were used to calculate the size of the interpolated twitch, by subtraction of the mean pre-stimulus force from the maximal post-stimulus force.

Voluntary Activation = = [1 - (Size of interpolated twitch / Size of resting twitch)] x 100

During familiarization sessions, participants alternated between performing MVCs with and without twitch interpolation, so that approximately three trials of each were performed within each session. Standardized strong verbal encouragement during each familiarization session/trial and real-time visual feedback of their performance was provided to the participants via the computer display onto a large screen placed in front of them.

iii) Back squat and bench press Participants were familiarized with back squat and bench press three times prior to commencement of the testing cycle. Each participant was asked to perform the back squat with incremental loads (40, 60, 80% 1RM) for one repetition at each load and 5-min rest was allowed between each effort. Likewise for bench press, each participant performed one repetition against each incremental load (40, 60 and 80 % 1RM) and again 5-min rest was given between each working effort. This was done so that the upper loads required for the experimental trials were known to be comfortably within each of the participants' physical capabilities and as such there was minimal likelihood of them failing to perform the required efforts for data collection. Each of the participants performed the two exercises in same order: bench press and then back squat, performing three lifts at each exercise against progressively increasing loads (40, 60 and 80 % 1RM). The individual's own body mass was factored into the back squat exercise, as this is a whole-body movement, but not into the bench press. The MuscleLab force-velocity linear encoder (Muscle Lab, Ergotest version 4010, Norway) was attached to an Eleiko Olympic bar (20 kg) which was set upon rests on a standard squat rack, safety arms were set so that the participant achieved ≤90° knee flexion position (settings measured and recorded during the familiarization process). From this position, the participant was instructed to drive the bar upwards as forcefully as possible; the value recorded during the test was for the concentric phase of the action only. The MuscleLab system encodes the force, the displacement of the bar, and the velocity produced for each individual lift, so that following measurements of muscle performance can be measured: average velocity (AV), average power (AP, average force x average velocity), peak velocity (PV, highest value of velocity), time to peak velocity (tPV, time to reach the highest value of velocity), mean propulsive velocity (MPV, average velocity during the propulsive phase of the concentric movement), rate of development of velocity (RDV, average acceleration during the propulsive phase of the concentric movement) and displacement (D, linear dis-placement of the bar during the movement). This process was repeated three times, with 5-min rest allowed between each individual lift, and against the progressive workloads as described above. For the bench press, the participant was instructed to descend the bar to their chest and, again, the instruction given was to push against the bar as forcefully as possible. This was repeated three times against the workloads described above, with 5-min rest between each push.

The highest of the three AP outputs (and associated AV, PV, MPV, RDV, D and tPV values) were used for analysis for each mass on the bar for both bench press and back squat, respectively. To reduce the likelihood of injury, two people were positioned either side of the participants as they performed their lifts, to intervene at any time if needed. In addition, there was safety support in place on every occasion.

Experimental Protocol and Measurements

The experimental sessions took place a week after the last familiarization with 72 h recovery between the three experimental conditions (i.e. CAFF, PLAC, NoPill). Upon arrival at the laboratory, they were asked to insert a soft flexi-ble rectal probe (mini-thermistor, Grant Instruments, Shepreth, UK) approximately 10 cm beyond the external anal sphincter. Participants then rested for 30 min in seated position to assess resting rectal temperature (Trec). Skin temperature (Tsk) was assessed simultaneously by skin thermistors (Grant Instruments, Squirrel 2010 series, Shepreth, UK), which were placed at four locations on the left side of the body (chest [ch], forearm [f], thigh [th] and calf [ca]). The average of the last 5 min of the 30 min resting period was recorded for resting Trec and Tsk temperatures. Mean Tsk was calculated as follows:

Tsk = (0.34 x Tch) + (0.33 x Tth) + (0.18 x Tca) + (0.15 x Tf)

The mean body temperature (Tmb) was calculated using:

Tmb = (0.64 x Tr) + (0.36 x Tsk).

Participants completed profile of mood states questionnaire (POMS; Version 32), their subjective rating of sleep (using sleep questions from the Liverpool Jet lag Questionnaire, effort (0-10, 0 = no effort and 10 = maximal effort) and rating of perceived exertion (RPE). After the initial rest measurements, participants warmed up on a cycle ergometer (Corival cpet, Lode, Groningen, Netherlands) for 5 min at 150 W. Post warm-up, participants removed rectal probes in private and skin thermistors were taken off. After which, MVC and bench press and back squat exercises were then undertaken.

Statistical analysis The main research variables in the present study were peak force in the MVC and PV in the bench press and back squat. For MVC peak force sixteen participants were estimated to provide 80% statistical power (p < 0.05), diurnal variation effect size (ES) of 0.67 for one tailed t-test. For PV for bench press nine participants were estimated to provide 80% statistical power (p < 0.05), diurnal variation effect size (ES) of 0.92 for one tailed t-test. For PV for back squat nine participants were estimated to provide 80% statistical power (p < 0.05), diurnal variation effect size (ES) of 0.91 for one tailed t-test. The investigators recruited 18 participants to allow for a dropout, with n=4 unable to complete the full protocol. The sample size estimation was performed using software G*Power 3.1. Data were analysed using the Statistical Package for Social Sciences version 30 (SPSS, Chicago, IL, USA). All data were checked for normality using the Shapiro-Wilk test. General linear models were used to analyse all measurements collected; the significance of interactions and main effects were evaluated by ANOVA. Sphericity violations were corrected by Greenhouse-Geisser (ε < 0.75) or Huynh-Feldt (ε > 0.75) and main effects by Bonferroni pairwise comparisons. Pearson correlations were conducted to explore individualized responses to CAFF vs other conditions (relative to body mass) and % change in MVC performance. All data is presented as means ± standard deviation (SD), unless otherwise stated. Significance was set at p ≤ 0.05. P values between 0.05 and 0.10 were considered to indicate a statistical trend. Effect sizes are referred to as partial eta squared values (η2p) with values of 0.01, 0.06 and 0.14 corresponding to a small, medium and large effect respectively. Ninety-five percent confidence intervals (95% CI) and mean differences between pairwise comparisons are reported where suitable.