2.2. Graded Exercise Test (GXT) and Familiarization

Peak aerobic power (VO_2peak) was assessed during a symptom-limited GXT on a cycle-ergometer (Excalibur sport, Lode B. V., Groningen, The Netherlands). The protocol consisted of an initial intensity of 60 W, then an increase of 30 W every 2 min. The test was terminated when a participant’s rating of perceived exertion (RPE) reached “very hard” (Borg scale = 17) or before that if the patient wished to stop or clinical signs or symptoms of metabolic or cardiorespiratory abnormalities appeared. Expired respiratory gases were collected through a breath-by-breath (BxB) pneumotach system connected to gas analyzers (Cosmed Quark CPET, COSMED, Rome, Italy). The BxB data were integrated over each 15 s interval, and the mean values for oxygen consumption (VO₂), carbon dioxide generation (VCO₂), and ventilation (VE) were calculated for each interval. The gas analyzers were calibrated immediately before each test as per the manufacturer’s instructions.

Dietary intake was controlled for the 3 days prior to the initial GXT via the provision of all food for the participants. Energy requirements for each participant were estimated using the Schofield equation and adjusted for physical activity level, and food intake was tailored to the client’s likes/dislikes such that an energy balanced diet was consumed that met the Australian guide to Healthy Eating (Foodworks, Version 10, Xyris Software, Brisbane, Australia 2019). Diets were designed by a qualified dietitian.

A familiarization of the TtE and TT within a week of the GXT was undertaken to ensure exercise intensities estimated from the GXT were appropriate and allowed the participants to become familiar with the exercise tasks to reduce daily test–retest variability between trials. During the familiarization of the SS and TtE, participants cycled at the SS power output determined by the GXT to elicit an intensity of 90% of ventilatory threshold for 10 min to verify that the power output was indeed below the ventilatory threshold. The work rate was then increased to a power output at 50% of the ventilatory threshold and the VO₂ peak until exhaustion, which was then repeated following 30 min of rest. Two days following the SS and TtE familiarization session, participants undertook one familiarization of the 20 km TT. The participants were familiarized with the distance of the time trial and the Velotron™ cycle ergometers (SRAM LLC, Chicago, IL, USA). This familiarization has previously been shown to be sufficient to lower variability from 3% to less than 1% [23].

Two weeks following the familiarization testing, the participants started with either placebo or the G. pentaphyllum supplement (randomized and double blinded) for 4 weeks and then undertook the SS + TtE and TT, separated by two days (Figure 1). The participants then completed a four-week washout period before repeating the supplementation and exercise testing regimen with the alternative supplement (i.e., placebo or G. pentaphyllum) not used in the first trial (Figure 1).