Initial screening

Prior to participation, volunteers were required to provide informed consent and complete an unpublished caffeine consumption questionnaire that has previously been employed to assess daily level of caffeine intake (Haskell et al. 2005). Volunteers were recruited to take part in the study if they fell into one of two pre-defined categories: ‘habitual consumers’ (those who drank tea and consumed more than 150 mg caffeine per day) or ‘non-habitual consumers’ (those who consumed less than 60 mg caffeine per day and no more than 2 cups of tea/coffee per week). These cutoffs are designed to allow the consumption of a 330 ml can of most standard colas per day in non-habitual consumers and to ensure that habitual consumers ingested the equivalent of at least three 190 ml cups of tea per day (Gray 1998). Only non-smoking volunteers who were in good health, not currently taking any dietary supplements or medication (including the contraceptive pill), were not colour-blind and did not have a history of head trauma, learning difficulties, ADHD, neurological, vascular or psychiatric illness were recruited to take part in the study.

Design

A double-blind, counter-balanced, within-subjects, placebo-controlled design was utilised.

Treatment

Participants attended four study visits, at least 48 h apart and, at each, received one of the following treatments: 75 mg caffeine (pharmaceutical grade caffeine powder, Blackburn Distributions Ltd.), 50 mg L-theanine (Suntheanine, Taiyo Europe, Germany), 75 mg caffeine and 50 mg of L-theanine in combination, or placebo. The doses selected roughly equate to the levels found in two cups of tea. These were chosen in an attempt to extend previous findings exploring the effects of 100 mg L-theanine (Einother et al. 2010; Kelly et al. 2008; Owen et al. 2008) whilst more closely reflecting the ratio of L-theanine to caffeine found in tea. Each treatment was administered in the form of two capsules in order to mask any taste differences and to ensure that participants remained blind to the treatment they had received. The capsules were prepared and coded by an independent third party who had no further involvement with the study. The order in which participants received each treatment was determined by Latin square and random allocation to treatment order for each group (habitual consumers and non-habitual consumers).

Participants

Twenty-four healthy young participants (10 males) between the ages of 18 and 35 (mean age 21.8, standard deviation (SD) 3.19) were recruited. Twelve participants were classified as habitual consumers (five males; mean age 23.3, SD 3.65) and 12 as non-habitual consumers (five males; mean age 20.4, SD 1.88). From the self-report caffeine consumption questionnaire, habitual consumers reported drinking between 163 and 432 mg caffeine per day (mean 252.2, SD 74.3). Non-habitual consumers reported drinking between 0 and 56 mg caffeine per day (mean 16.7, SD 15.6). With regards to tea consumption, habitual consumers reported consuming between 1 and 6 cups per day (mean 3.50, SD 1.46) and non-habitual consumers reported consuming between 0 and 2 per week (mean 0.45, SD 0.62). The study was approved by Northumbria University’s School of Psychology and Sport Sciences’ ethics committee and was conducted according to the Declaration of Helsinki (1964).

Salivary caffeine levels

Saliva samples were obtained using salivettes (Sarstedt Ltd). One sample was taken upon arrival and one immediately following the post-dose cognitive assessment. This was to ensure overnight caffeine abstinence and to confirm caffeine absorption following caffeinated treatments (no analysis of post-treatment caffeine levels was made following placebo or L-theanine). Once taken, samples were frozen at −20 °C. The samples were then thawed and the caffeine levels in the saliva samples were measured using an Emit® Caffeine Assay (Dade Behring Ltd).

Cognitive and mood assessment

All cognitive and mood measures were delivered using the Computerised Mental Performance Assessment System (COMPASS, Northumbria University, Newcastle upon Tyne, UK), a purpose-designed software application for the flexible delivery of randomly generated parallel versions of standard and novel cognitive assessment tasks. This assessment system has previously been shown to be sensitive to nutritional interventions (Kennedy et al. 2010a; Stonehouse et al. 2013) including caffeine (Kennedy and Haskell 2011). The tasks were chosen based on their known sensitivity to one or both of the nutritional interventions under investigation (Haskell et al. 2008; Kennedy and Haskell 2011; Lieberman et al. 1987) and, in order to correspond with the region of CBF measurements, their ability to activate the pre-frontal cortex (Drummond et al. 1999; Lawrence et al. 2002; Schroeter et al. 2002). The tasks completed at baseline and post-dose were identical with the exception that baseline tasks were shortened to 2 min. This ensured that participants were not connected to NIRS equipment for more than 2 h and therefore minimised the discomfort associated with restricted movement. Tasks were presented in the following order (post-dose duration in parentheses): serial 3 subtractions (4 min), serial 7 subtractions (4 min), simple reaction time (SRT) (8 min), rapid visual information processing (RVIP) (8 min), choice reaction time (CRT) (8 min) and Stroop (8 min).

Serial 3 subtractions

A starting number between 800 and 999 appears on the screen and participants are instructed to count backwards as quickly and as accurately as possible from this number in threes, using the linear number keys to make their response. Responses are cleared when the ‘enter’ key is pressed. Participants are only shown one number on screen and the rest of the numbers are generated by subtracting from the previous number in their head. In the case of incorrect responses, subsequent responses are scored positively if they are correct in relation to the new number. This timed task was scored for total responses and number of errors.

Serial 7 subtractions

This task is identical to the serial 3s subtraction task except that it involves the serial subtraction of 7s.

Simple reaction time

An upwards pointing arrow appears on the screen at a random inter-stimulus duration between 1 and 3.5 s. Participants have to respond as quickly as they can when they see a stimulus appear by pressing the space bar. One hundred and ninety stimuli were presented and the task was scored for reaction time. Responses below 150 ms do not register for the task.

Rapid visual information processing

The participant monitors a continuous series of digits for targets of three consecutive odd or three consecutive even digits. The digits are presented on the computer screen at the rate of 100 per minute in pseudo-random order, and the participant responds to the detection of a target string by pressing the space bar as quickly as possible. The task is continuous. The task was scored for percentage of target strings correctly detected, average reaction time for correct detections, and number of false alarms.

Choice reaction time

An arrow pointing either left or right is presented in the middle of the screen at a random inter-stimulus duration between 1 to 3.5 s. As soon as participants see an arrow appear on the screen, they are required to indicate the direction of the arrow by pressing left and right keys (‘M’ and ‘Z’ on keyboard). One hundred and eighty-five stimuli were presented and the task was scored for percentage of correct responses and reaction time. Responses below 150 ms do not register.

Stroop

In this task, a series of colour names appear on the screen one at a time in different coloured fonts. Participants are required to use a colour-coded response pad to select the colour that matches the colour font that the word is written in. The words that are presented are either ‘congruent’ (name of colour and colour of ink the same) or ‘incongruent’ (name of colour and colour of ink different) and are presented randomly. Participants are asked to respond as quickly and as accurately as possible. This timed task was scored for total responses, number of errors, reaction time and interference reaction time (difference in reaction time for congruent and incongruent stimuli). Responses below 150 ms do not register.

Subjective assessment

Caffeine research visual analogue scales adapted from Rogers et al. (2003) that have previously been used in caffeine and L-theanine research were also included (Haskell et al. 2005, 2008; Kennedy and Haskell 2011) and were presented on-screen. Participants were shown the following descriptors ‘relaxed’, ‘alert’, ‘jittery’, ‘tired’, ‘tense’, ‘headache’, ‘overall mood’ and ‘mentally fatigued’ and asked to rate how much they matched their current state by placing an ‘x’ on a 100-mm line with the end points labelled ‘not at all’ (left hand end) and ‘extremely’ (right hand end), with the exception of ‘overall mood’, which was labelled ‘very bad’ and ‘very good’. ‘Alert’ and ‘tired’, and ‘tense’ and ‘relaxed’ scores were then combined to create respective factors of ‘alertness’ and ‘tension’ as recommended by the authors.

Near-infrared spectroscopy measurements

Near infrared spectroscopy (NIRS) is a method of studying functional activation through monitoring changes in the haemodynamic properties of the brain (Huppert et al. 2006). It is a non-invasive brain imaging technique in which two wavelengths of light that are differentially absorbed by oxygenated and deoxygenated haemoglobin are introduced through the skull via a laser emitter and measured following transit through the upper surface of the cortex, by an optode placed at a pre-set distance from the light source.

Relative changes in the absorption of near-infrared light were measured at a time resolution of 10 Hz using a 12-channel Oxymon system (Artinis Medical Systems B.V.). The system emitted two nominal wavelengths of light (~780 and 855 nm) with an emitter/optode separation distance of 4 cm. The differential path length factor was adjusted according to the age of the participant. Relative concentration changes in oxygenated haemoglobin (oxy-Hb), deoxygenated haemoglobin (deoxy-Hb) and total haemoglobin (total-Hb) were calculated by means of a modified Beer–Lambert law using the proprietorial software. In this study, a simple two emitter/receiver optode pair configuration was utilised (i.e. two channels). The emitter/receiver optode pairs were positioned over the left and right pre-frontal cortex that included the areas corresponding to the international 10-20 system Fp1 and Fp2 EEG positions. The ability of NIRS to measure blood flow changes following cerebral activation has been validated by its use in a number of studies (Fallgatter and Strik 1998; Izzetoglu et al. 2003, 2004; Schroeter et al. 2002; Shibuya-Tayoshi et al. 2007) including following different nutritional interventions (Jackson et al. 2012a, b; Kennedy and Haskell 2011; Kennedy et al. 2010b; Wightman et al. 2014).

Blood pressure and heart rate

Blood pressure and heart rate (Boso-Medicus Prestige; Bosch + Sohn, Germany) readings were taken from the left arm following a 5 min seated rest. Readings were taken at each visit upon arrival and following post-dose completion of the cognitive tasks.

Procedure

Participants were required to attend the laboratory on five separate occasions. The first visit was a screening session where participants were informed about the nature of the study, its requirements and its restrictions. Written informed consent was obtained and their eligibility to participate was confirmed. Habitual caffeine intake and source were assessed via questionnaire, and familiarisation with the tasks to be administered on the study days was conducted. The remaining four study visits were identical to each other, with the exception of the treatment administered. On each day, participants attended the lab at 8 a.m. following an overnight 12-h fast during which they were only permitted to drink water. Upon arrival, heart rate and blood pressure readings were taken following 5 min of seated rest. Following a baseline completion of the mood scales, salivary caffeine levels were taken to ensure caffeine abstinence. Following this, the NIRS headband was fitted. Participants initially sat quietly for 5 min and then made a baseline completion of the cognitive tasks. Upon completion of the tasks, participants were required to sit quietly for a 2 min NIRS resting baseline period. Participants were then required to take their treatment for the day. Following a 30 min absorption period (during which time NIRS recording continued whilst participants watched a non-stimulating wildlife DVD), participants completed a second set of the cognitive tasks and a final rest period (8 min) (included to allow an assessment of whether any CBF effects apparent after the absorption period are as a result of increased neural demand during tasks or are simply due to the time course of effects of treatment). They then had their blood pressure and heart rate measured for a second time, rated their mood for a second time, and gave a second saliva sample, used to confirm caffeine absorption following caffeinated treatment (see Fig. 1 for more details of procedure and task duration). Participants returned for their next study visit within 7 days, following (at least) a 48-h washout period.

Fig. 1 Timeline representing flow of study day Full size image

Statistics

To assess the possibility of any on-day or consumer status differences in cognitive performance, mood, blood pressure and heart rate prior to treatment, two-way repeated measures ANOVAs were conducted (treatment × consumer status) on baseline data. Any significant differences were further explored with Bonferroni-corrected pairwise comparisons.

In order to confirm caffeine absorption for caffeine-containing treatments (caffeine, combination), a two-way mixed ANOVA was conducted (treatment × time) on pre- and post-caffeine and combination saliva samples.

NIRS data was averaged across 4 min (absorption period) and 8 min (individual tasks) epochs and baseline adjusted to the 2 min post-task resting pre-treatment period. To account for disruption in NIRS readings as a result of consumption of treatment, the first 2 min of the absorption period was omitted from the analysis. Data from both channels were averaged for oxy-Hb and deoxy-Hb and analysed by three-way mixed ANOVA (epoch × treatment × consumer status). Significant treatment-related interactions were further investigated by a priori planned comparisons where each active treatment was compared to placebo at each epoch utilising t tests calculated with the mean squares error from the ANOVA (Keppel 1991). In order to reduce the potential for type I errors, only those planned comparisons associated with a statistically significant difference on the initial ANOVA are reported.

Cognitive performance, subjective mood, heart rate and blood pressure data were analysed using the MIXED procedure in SPSS 21.0. The model included the respective baseline as a time variant covariate, and fixed effects terms entered into the model were treatment, consumer, treatment*consumer and baseline value. Significant effects or interactions (p < 0.05) were further explored with Bonferroni-corrected pairwise comparisons.