Participants

Following Institutional approval nine moderately trained males (mean ± SD; Age 21 ± 1 years, body mass 76.0 ± 9.3 kg, stature 177 ± 9.4 cm and \( \overset{.}{V}{O}_{2 max} \) 41.89 ± 5.4 ml/kg/min) volunteered to participate and provided written informed consent to participate in the study. All procedures and conduct met with Declaration of Helsinki.

Dietary control

Participants were instructed to maintain their normal diet and refrain from alcohol, vitamin supplements, anti-inflammatory products and from consumption of milk two hours before and after consumption of either supplementation, to increase absorption over the period of intervention. Participants were also provided with a dietary list of prohibitory foods high in nitrate with an appropriate low nitrate replacement. To prevent an increase surplus in daily caloric energy intake, participants replaced a snack meal or dessert with similar caloric values for DC/WC and were instructed not reduce their fruit and vegetable consumption. The DC and WC were best matched for calorific content (total energy provision over 14 days was 12887 kJ & 12945 kJ for DC and WC respectively) and no other supplementation was allowed throughout the intervention including consuming any other chocolate. In the 24 h preceding the first exercise test, participants were asked to record their dietary intake, detailing all foods consumed, and this diet was replicated in the 24 h preceding subsequent tests. Participants were instructed to avoid any strenuous activity in the 24 h preceding each testing session, to refrain from caffeine for six hours and from alcohol for 24 h before each testing session. During the 2-week washout period between interventions, participants were not required to adhere to any specific dietary regime, but to avoid chocolate.

Experimental design

To examine whether dark chocolate consumption affects \( \overset{.}{V}{O}_{2 max} \) (ml/kg/min), blood pressure (BP) (mmHg), oxygen cost (ml/kg/min), and lactate levels (mmol/L) during a 20-min cycle test at 80 % GET and all out sprint performance (m), a randomised crossover design was used with participants blinded to the aims of the study. Baseline tests (BL) were used to accustom participants to testing protocols (Fig. 1), establish GET for the entirety of the protocol, and for participants to be randomly assigned to either a daily intake of DC (40 g, DOVE®, Dark Chocolate, Mars, Incorporated, Hackettstown, NJ) or white chocolate (WC) (40 g of Milkybar®) for fourteen days, followed by a seven day wash out period, then switched to the alternative treatment. The commercially available dark chocolate was chosen as it was previously reported to be rich in (-)-epicatechin [14]. Fourteen days of supplementation was used as Sudarma et al. [13] reported increases in NO following DC consumption of this quantity an duration.

Fig. 1 Schematic illustration of the exercise test protocol Full size image

Equipment and procedures

All three laboratory visits were performed at the same time of day on different days and performed in a standardised order (Fig. 1).

Resting measures

Stature was measured to the nearest 0.1 cm using stadiometer and body mass was measured to the nearest 0.05 kg using an electronic scales (Seca, Vogel & Halke, Germany). Participants were rested supine for five minutes before BP measurements were taken using a Maruman digital automatic BP monitor (Maruman Co. Ltd., Tokyo, Japan). On each occasion, three measurements were taken, with a 60s rest between each measurement and the mean value reported.

Incremental test \( \left(\overset{.}{V}{O}_{2 max}\right) \)

All tests were performed on an electromagnetically braked cycle ergometer (Velotron Cycles, Racermate Inc., Seattle, Washington, USA). A ramp incremental test was used in accordance with Bailey et al. [1] procedures with the primary purpose to establish each participant GET. Participants cycled at a self-selected pedal rate between 70–90 rpm for 3-min ‘unloaded’ (0 W) warm up, after which the work rate was increased at a rate of 30 W/min until volitional exhaustion. Pedal rate, saddle height, and handle bar height configuration were recorded and reproduced in subsequent tests. Pulmonary gas exchange was continually collected throughout the incremental test (Oxycon Pro, VIASYS GmbH, Eric Jaeger, Hoechberg, Germany) and was averaged to fifteen seconds collection periods. \( \overset{.}{V}{O}_{2 max} \) was recorded as the highest value achieved over a 15-s collection period.

Moderate intensity cycle

Participants rested for 30 min after completing the incremental test, before commencing moderate cycling. To ensure consistency across conditions, the GET and corresponding power output for all three conditions (BL, DC, WC) was calculated from the BL \( \overset{.}{V}{O}_{2 max} \) test. The GET from the baseline test was determined as the work rate corresponding at the precise cross over, at which V E / \( \overset{.}{V}{O}_2 \) continually exceed V E /VCO 2 . A power output corresponding to 80 % of GET was used as the moderate cycling intensity for twenty minutes at a self-selected pedal cadence. Heart rate and pulmonary gas exchange were collected continually throughout the 20-min cycle and averaged to five-minute intervals. Capillary blood was collected from the index fingertip every five minutes and analysed for concentrations of lactate (Arkray Factory Inc, 1480 Oaza Kouji umeda, kounan-cho kouka gmn, Shiga, Japan).

Distance time trial

On completion of the moderate intensity cycling, participants were instructed to cycle maximally for two minutes and the total distance (m) achieved was recorded.

Statistical analysis

Data was analysed for normality using Shapiro Wilks. A two factor within-subject repeated measures ANOVA was used to analyse the differences over the 20 min of moderate intensity exercise (oxygen consumption, RER, lactate levels and heart rate) with Newman Keuls post-hoc tests used when differences were found. A paired t-test was used to measure the difference between all other dependent variables. Data was presented as mean ± standard deviation (SD), unless otherwise stated. Statistical significance was accepted when P ≤ 0.05.