The goal of this clinical trial is to test whether changes in lactate kinetics during exercise (due to glycogen depletion or hyperthermia) alter the pattern of fat oxidation during a maximal incremental cycle ergometer test in healthy young active people. The main questions it aims to answer are:

• Will a rightward shift in lactate kinetics, induced by a previous glycogen depletion, produce a rightward shift in fat oxidation?
• Will a leftward shift in lactate kinetics due to an increase in ambient temperature produce a leftward shift in fat oxidation?

Participants will perform three maximal incremental tests in three different conditions:

• one in the control condition;
• one with glycogen depletion;
• and one with ambient heat (the latter two in randomized, counterbalanced order).

Introduction Lactate has three major functions: it is an energy substrate, it is a gluconeogenic precursor, and it is a signalling molecule (1). Among the signalling functions is that of modulating skeletal muscle metabolism, as a regulator of fat oxidation at both the autocrine and paracrine levels. At the autocrine level, lactate increases the levels of Acetyl-coenzyme A (CoA) inside the mitochondria and, consequently, the levels of Malonyl-CoA. The increase in Malonyl-CoA concentration inhibits Carnitine PalmitoylTransferase 1 (CPT1), which is one of the transporters of fatty acids into the mitochondria (2). At the paracrine level, an increase in blood lactate concentration reduces the release of fatty acids into the blood by adipose tissue (3). This occurs because the G protein-coupled receptor present in adipose tissue inhibits the release of fatty acids into the blood when the blood lactate concentration increases (4). However, in vivo, only an inverse correlation between blood lactate concentration and fatty acid oxidation has been demonstrated (5,6). Therefore, the main objective of the study is to observe whether the pattern of fat oxidation during a maximal incremental cycle ergometer trial is altered due to changes in lactate kinetics during the given trial. For this purpose, three maximal trials will be performed with the same loading protocol (intensity and progression of the same), but different physiological conditions (normal vs. previous glycogen depletion vs. environmental hyperthermia).

Procedures Consent to participate (signature of informed consent) will be requested from all volunteers who meet the inclusion criteria.

The tests that will be performed throughout this study will be the following:

1. At the first appointment, you will be asked about your medical history. Then, if appropriate, an assessment of your body composition by dual X-ray absorptiometry (DEXA) will be performed,
2. In the second appointment, spirometry, an electrocardiogram at rest, and finally, an ergospirometric trial will be performed.
3. In the third, fourth, and fifth visits, three maximal exercise trials will be performed, one each day. Of the three maximum ergospirometric trials that will be performed, one will be in a hot environment -approximately 38º Celsius (ºC) ambient temperature- and the other in a state of glycogen depletion. A glycogen depletion protocol and a low-carbohydrate diet will be performed before the maximal ergospirometric trial in the glycogen-depleted state, while the other two trials (simple maximal trial and hot-environment maximal trial) will be performed after 24h of a high-carbohydrate diet.
4. The order of performance of each protocol will be randomized, provided that the glycogen depletion protocol is performed at the visit prior to the maximal ergospirometric trial under glycogen depletion. The depletion protocol consists of a 60-minute continuous cycle ergometer effort at the power associated with 60% of the maximal oxygen uptake (VO2) followed by six 30-second maximal efforts with four minutes of recovery. After this glycogen depletion exercise protocol, the participant will follow a low-carbohydrate diet (2% carbohydrate, 78% fat, and 20% protein), until the time of the ergospirometric trial under glycogen depletion status.

Inclusion Criteria:

• Perform endurance training with a volume of between 3 and 12h/week
• In women, having regular menstrual cycles (25-35 days length)
• In women, being able to perform the test without menstrual discomfort or pain and without having taken anti-inflammatory or analgesic drugs

Exclusion Criteria:

• Suffering from any previous disease, disorder or ailment that could affect the results of the study variables
• Smoking
• In women, using any type of hormonal contraceptive
• In women, having heavy menstrual bleeding, detected with the menstrual bleeding questionnaire proposed by Wyatt et al. (2001)

No Masking is possible since participants and investigators know which type of test (control, hyperthermia and glycogen depletion) is being performed.