All methods described here have been approved by the local ethical committee (Doshisha University Ethical Committee: 15033), and were in strict accordance with the standards set by the Declaration of Helsinki. Informed consent was obtained from all participants. Participants were male recreational athletes (age, 20.1 ± 1.2 years; weight, 69.7 ± 6.2 kg; height, 176.1 ± 5.8 cm; body fat 13.6 ± 1.5% and VO₂max, 52.6 ± 6.6 mL/min¹/kg¹).

1. Establishment of the Exercise Protocol

NOTE: The present study consisted of the evaluation of blood hormonal and metabolites dynamics by blood sampling from the antecubital vein during a HIIE protocol.

1. Ask the subject to perform a warm up with 5 min of cycling in the ergometer at low intensity. After the warm-up, let the subject rest for 5 min.
2. Ask the subject to perform the following HIIE in the leg cycle ergometer: three bouts of 30 s high-intensity exercise, separated by 4 min of rest (Figure 1).
   NOTE: The 30 s bouts should be set at an intensity equal to 90% the power average of a previously performed Wingate test. Refer elsewhere⁸ for a complete description of the Wingate test.
3. To calculate the workload for the 30 s bouts, use the following equations:
   Work = Force x Distance (equation 1)
   Force = Mass x Acceleration (equation 2)
   Work = (Mass x Acceleration) x Distance (equation 3)
   NOTE: In this model, the mass is the work load (kg), and the acceleration is 9.81 m/s². The distance is the wheel diameter (1.622 m). Because the wheel rotates 3.7 timed /peal rotation, the distance per pedal rotation is 3.7 x 1.662. Furthermore, because the pedal frequency is 90 rev/min¹, we have to multiply one pedal distance by 90.
   Work = Work load x 9.81 x 3.7 x 1.622 x 90 (equation 4)
   Work load = Work / (9.81 x 3.7 x 1.622 x 90) (equation 5)

2. Catheterization

NOTE: Tubes and vacutainers should be labeled beforehand to avoid losing time.

1. Wrap a tourniquet to the arm selected for the catheterization. To do this, place an elastic band at the back of the chosen arm. Stretching the band, make a loop with the band around the arm and tie the elastic.
2. Select the visible antecubital vein and sterilize the skin with ethanol or alcohol.
3. Insert the tip of the sterilized cannula into the vein at an approximate angle of 15° with the skin.
   NOTE: The cannula should be inserted from the proximal to distal direction. If cannulation is successful, a backflush of blood should be observed.
4. Secure the cannula with the hand, and remove the needle softly.
   NOTE: To maintain the angle of the needle during the exercise, cotton tissues may be used in between the skin and the needle. Fix this structure with a tape.
5. Connect a vacutainer with heparin and aspirate for the first blood sample (approximately 2.5 mL). Immediately after, refrigerate the blood sample by putting it in an ice box for no more than 40 minutes. Ask an assistant for help, if blood gases are going to be analyzed.
6. Connect a tube to the catheter and inject heparin to the full length of the tube so as to avoid blood coagulation. Close the butterfly.
7. Using tape, stabilize the catheter. Ensure that at this point, the arm mobility of the subject does not interfere with the blood collection. To confirm this condition, be sure that the needle position is not affected by any movement.

3. Blood Samplings

1. Remove the heparin by using a vacutainer until there is only blood in the catheter. When blood flashback is observed, collect the sample as described in step 2.5.
2. After the sample collection, inject heparin again into the catheter tube to avoid blood coagulation. After this action, step 2.5 may be repeated for several samples collection.
3. After the last sample is collected, carefully remove the cannulation tube and clean the skin with alcohol. To avoid possible bleeding, ask the subject to keep sterilized cotton at the place where the catheterization was made.
4. After blood is collected in the vacutainer tubes, carefully pass approximately 1.5 mL of blood to a 1.5 mL tube and keep the tubes on ice.
5. Measure blood lactate from the remaining blood using a lactate detector. On the same day, obtain the plasma the blood samples (around 1.5 mL of blood).
6. For plasma separation, centrifuge the blood samples for 5 min at 5000 x g at 4 ^oC, and separate the supernatant in a new 1.5 mL tube.
7. Repeat the centrifugation one more time to purify the plasma.
8. After the plasma separation, store the samples at -80 ^oC until the hormone analysis.
9. Measure the plasma insulin using an ELISA kit as per the manufacturer's protocol (see Table of Materials).