Diurnal Variation of Exercise on Metabolic Health

A continuous glucose monitor inserted into the skin of the arm upper forearm area will be used to record and store blood glucose levels every 15 minutes, for 24 hours, 14 consecutive days. Within this time range both exercise conditions will be performed. The monitor will be placed 24 hours before the first exercise session and will be removed 48 hours after the second exercise session. Therefore, change from resting blood glucose 24 hours previous to 48 hours after exercise will be obtained for each condition.

Participants will lay down for 60 minutes where resting gas exchange was measured (using a canopy hood for gases collection) for 30 minutes in two separated 15 minutes stages, corresponding to 45 to 60 and 75 to 90 minutes after exercise, respectively

Blood samples will be taken after exercise, immediately after exercise and 90 minutes after exercise, in order to measure change in plasma concentration of markers of glucose metabolism from resting to 90 minutes after exercise.

Blood samples will be taken to measure plasma concentration of markers of fatty acid metabolism before, immediately after and 90 minutes after exercise.

Muscular biopsies will be taken from the vastus lateralis' distal part of the quadriceps in a sub-sample before and immediately after the exercise. Biopsies will be performed by an experienced surgeon using microbiopsy needles (Achieve Automatic Needle 16G x 15 cm), obtaining ~30 mg per biopsy after previous local anaesthesia with 2% lidocaine. From each time point (Pre and Post), 1 skeletal muscle samples will be collected and immersed in liquid nitrogen and stored at -80ºC until further analysis (i.e.: transcriptomics). RNA from skeletal muscle will be extracted using Trizol (Invitrogen).

Muscular biopsies will be taken from the vastus lateralis' distal part of the quadriceps in a sub-sample before and immediately after the exercise. Biopsies will be performed by an experienced surgeon using microbiopsy needles (Achieve Automatic Needle 16G x 15 cm), obtaining ~30 mg per biopsy after previous local anaesthesia with 2% lidocaine. From each time point (Pre and Post), 1 skeletal muscle samples will be collected and immersed in liquid nitrogen and stored at -80ºC until further analysis (i.e.: proteomics).

Muscular biopsies will be taken from the vastus lateralis' distal part of the quadriceps in a sub-sample before and immediately after the exercise. Biopsies will be performed by an experienced surgeon using microbiopsy needles (Achieve Automatic Needle 16G x 15 cm), obtaining ~30 mg per biopsy after previous local anaesthesia with 2% lidocaine. From each time point (Pre and Post), 1 skeletal muscle samples will be collected and treated fresh to study mitochondrial respiration.

Muscular biopsies will be taken from the vastus lateralis' distal part of the quadriceps in a sub-sample before and immediately after the exercise. Biopsies will be performed by an experienced surgeon using microbiopsy needles (Achieve Automatic Needle 16G x 15 cm), obtaining ~30 mg per biopsy after previous local anaesthesia with 2% lidocaine. From each time point (Pre and Post), 1 skeletal muscle samples will be collected and immersed in liquid nitrogen and stored at -80ºC until further analysis (i.e.: mitochondrial protein supercomplexes by blue-native PAGE).

Measures of appetite before, immediately after and 90 minutes after exercise using an appetite visual analog scale (appetite VAS) scoring from 0 to 10, where 0 is the minimum punctuation for appetite and 10 is the maximum punctuation for appetite.

Before, during and after exercise, periodic recordings of the central and distal temperature will also be made by using thermal iButtons (iButtons DS 1922 L, Maxim, Dallas, USA).

Standarditation and control of diet. Diet will be standardized 24 hours prior to each exercise session (55% carbohydrates, 27% fat and 18% proteins). In addition, diet will be monitored for 48 hours after the exercise test.

Levels of physical activity will be controlled by accelerometry 24 hours before and 48 hours after each exercise session (Actigraph, GT3X).

Body fat percentage will be evaluated by dual X-ray absorptiometry (Discovery Wi, Hologic, Inc, Bedford, MA, USA).

Weight and height will be measured (Seca model 799, Electronic Column Scale, Hamburg, Germany). Weight and height measurements will be aggregated to obtain BMI in kg/m^2

Participants will complete the HÖME questionnaire, that determines chronotype (morning-evening).

Dates of the different phases of the menstrual cycle will be recorded in order to locate all tests in the luteal phase. However, we have observed that fat oxidation is not affected by the phase of the menstrual cycle