3.1.2. Mitochondrial Markers

A key feature of the adaptive response to endurance training are changes in the activity of enzymes involved in the tricarboxylic acid (TCA) cycle and the β-oxidative pathway [154]. Activity of citrate synthase (CS) is the most widely used biomarker of mitochondrial content in skeletal muscle because of the strong correlation between resting CS activity and resting mitochondrial content when measured using the "gold standard" transmission electron microscopy (TEM) [155]. Similar changes in CS activity have been observed between fasted and fed-state training following 4–6 weeks of moderate-intensity training [12,13] and HIIT [143,146]. A key enzyme of the β-oxidative pathway, β-hydroxyacyl coenzyme A dehydrogenase (β-HAD), is also generally not impacted by pre-exercise nutrition [12,13,143]. However, one study has shown an increase in both CS and β-HAD only with fasted, but not CHO-fed training [145]. It is possible that this difference may be related to the very large amount of CHO ingested in the fed-training group (~2 g kg−<sup>1</sup> 90 min prior and 1 g kg−<sup>1</sup> h−<sup>1</sup> during exercise), as other studies showing similar adaptations between fed and fasted training used smaller (e.g., 1–1.5 g kg−<sup>1</sup> CHO) pre-training meals [13,143]. Increases in succinate dehydrogenase activity following twice-daily training were blunted when ingesting CHO before and during the second workout, which was commenced with lowered muscle glycogen [93], suggesting a strong, and potentially underappreciated influence of ingesting CHO during exercise that adds complexity when interpreting the current literature.

Greater increases in CS have been reported in two studies that had subjects train twice-daily every other day, inducing low muscle glycogen during the second bout of exercise, compared with once-daily training with normal muscle glycogen [35,150]. In these studies, the two sessions were 1–2 h apart and subjects received only water between sessions. In contrast, other studies using twice-daily training but feeding low- or high-CHO meals between sessions found similar training-induced increases in CS activity between groups [151,153]. When comparing two different "train-low" protocols (2 h vs. 15 h between low-glycogen training sessions), greater elevations in acute signaling and mitochondrial adaptations were observed when training with 2 h between sessions without ingesting any food [152,156]. Thus, it appears that remaining in the fasted state following the first bout of exercise may be an important factor in the augmented adaptations observed following twice-daily training.

Overall, the exercise training itself seems to be the primary driver of changes in mitochondrial content, though very large pre-exercise meals (>1.5 g/kg CHO) and CHO ingestion during exercise may have blunting effects on some signaling pathways, possibly related to the interactions between AMPK and glycogen [79]. Future research should explore the effects of pre-exercise nutrition choices on contraction-induced and RONS signaling pathways.

#### 3.1.3. VO2max and Peak Aerobic Power

Studies comparing fasted and fed training have reported no differences in VO2max following 4 weeks of sprint interval training (SIT) [144], 6 weeks of aerobic training [14,145], and 3 weeks of mixed

intensity training [157]. However, greater training-induced increases in VO2max have also been reported following both fasted vs. fed-state training [13] and fed vs. fasted-state training [12]. Reasons for these divergent findings are unclear, as both studies used untrained participants performing 4–6 weeks of steady-state aerobic training. Similar improvements in VO2max and peak power were seen in untrained men following 8 weeks of HIIT with or without prior CHO [146], and following exercise undertaken with low or high muscle glycogen levels in trained and untrained athletes [35,93,153,158,159].
