*4.1. Overview of Study Methods*

Twelve competitive male cyclists participated in this study, which was approved by the human research ethics committees of the Australian Institute of Sport (20140612) and the Australian Catholic University (2014 254N). These subjects (32.6 ± 5.1 years; 79.2 ± 9.5 kg; 5.1 ± 0.6 L/min maximum oxygen consumption, and 639 ± 115 W maximum power output) represented a sub-group of a larger cohort who undertook the main project under

which this study was performed [28]. This study employed a parallel group design to investigate the effect of creatine loading, followed by a within-group cross-over application of carbohydrate loading on muscle substrate, water content, and performance (Figure 1). The participants came in for four separate biopsy and ultrasound measurements; baseline (day 0, 6 g carbohydrate/kg body mass (BM)/day for 48 h), glycogen depleted (day 1) and either glycogen loaded or glycogen normal (6 g carbohydrate /kg BM/d for 48 h) with or without creatine supplementation (days 7 and 14). Manipulations of creatine and glycogen stores were achieved by implementing "best practice protocols" of creatine loading (20 g/day for 5 days loading and 3 g/day for maintenance) [30] and glycogen loading (12 g CHO/kg BM/day for 48 h) [31] through a standardised pre-packaged diet. Furthermore, a supervised cycling protocol of ~3.5 h was undertaken to deplete muscle glycogen stores. The cycling protocol involved a 120 km time trial, with alternating 1 km and 4 km sprints every 10 km, followed by a ride to exhaustion at 8% gradient and 88% VO2max; further details can be found in [28]. Participants consumed 60 g/h CHO during the cycling protocol with post-exercise intake of a low CHO diet (<1 g CHO/kg BM) to restrict the repletion of glycogen stores before a further assessment of muscle glycogen content the following morning. This protocol was chosen to allow us to align our assessment of the depleted glycogen condition with DXA-estimates of body composition assessed according to best practice protocols (overnight fasted and rested conditions [32]). This study design provided situations where muscle glycogen was measured under baseline and normalised conditions, a depleted condition, and CHO loaded with or without creatine loading. Four biopsies were conducted over the course of the study, with each being collected from the same leg from an incision that was as least 2 cm from the previously biopsied site [19] [Figure 1: Bx1–Bx4]. The protocol used for these biopsies, and the determination of glycogen and creatine content in the muscle samples, is described in full elsewhere [28].

**Figure 1.** Overview of study design. (TT, time trial; RTE, ride to exhaustion; ultrasound; and Bx, biopsy).
