Subjects

Before the study could begin, the researchers conducting it had to find twelve people to be their "guinea pigs." They couldn't be just any twelve people, either. The volunteers needed to meet specific requirements. Six needed to be healthy endurance-trained cyclists. The other six had to be healthy too, but they couldn't have any previous cycle training. All the participants had to be males to ensure consistent results. Finally, all they had to be ready to do some serious exercising!

Preliminaries

About 3 weeks before the study took place, all twelve participants had to come in for some prelinary testing. First, each participant's VO_{2 peak} needed to be determined. A VO_{2 peak} is the maximal rate of oxygen utilized by the entire body. Then, the exercising began! Another test that would help compare and interpret results determined the weight and percent body fat of each man.

Diet

The participants had to eat certain diet before the study, starting 2 days beforehand. They had to eat a certain amount of calories and their diet had to be 50% carbohydrates, 30% fat, and 20% protein. This diet would promote fast build-up of muscle glycogen storage. The night before the actual study tests began, the participants had to fast. That's right, no midnight snacks for them! This would ensure that all the blood readings accurately reflected the levels resulting from the test and not something the men ate beforehand. After the study exercise tests, the subjects also had to eat a controlled diet at specific times for 72 hours.

Test Procedure

Each of the 12 men cycled on an ergometer so that each one worked at 75% of their individual VO_{2 peak} for 2 hours. (An ergometer is like an exercise bike without wheels. It is used by astronauts for exercise while in space.) They were allowed to take a 4-minute break every half hour. After cycling, each one had to do 1 minute sprints at 100% VO_{2 peak}. Each subject did 5 sprints, resting 3 minutes between each one. Sounds exhausting! After these exercises, the 12 men were not allowed to exercise for 2-3 days.

Biopsies

Before the men started exercising, an incision was made in the thigh of each subject, right into the muscle. (Don't worry, they numbed it!) Directly after the subjects finished the exercises, a sample of the muscle was taken from the incision, then frozen to keep it in tact for analysis. Additional muscle samples were taken and tested at 6 hours after they began their first post-exercise meal. Ths was done yet again 48 hours after exercise on half of the subjects and at 72 hours for the other half. Muscle tissue was tested for Glut 4 protein, fiber type, and glycogen synthase. Only the first test was used to examine glycogen synthase.

Blood Tests

As if fasting and having incisions made in their legs weren't enough, the 12 subjects also had catheters inserted into their veins before the procedure started. Blood samples were taken and tested for various factors that either related to glycogen build-up or affected the results of the experiment.

Q: Why do consistent results depend on all subjects being male?
A: Men and women's bodies are physiologically different. They respond differently to exercise. In any science experiment, the test subjects must be as similar as possible to reduce the chance that differences in the subjects cause results to change. Therefore, to eliminate the possibility that physiological differences would cause different results, the scientists tested only one gender.

Q: What kinds of exercise did the subjects have to do?
A: All individuals performed strenuous cycling and running exercises. Each man cycled on an ergometer, an exercise bike without wheels and most likely ran on a treadmill.

Q: What did the subjects' special diet include and why did they have to eat it?
A: Two-thirds of the whole day's carbohydrates were consumed 6 hours after the test exercises. They ate at 0, 2, and 4 hours after exercise and their food choices were limited to healthy foods such as bagels and fruit and a drink with 75/100g of glucose at each meal. They had to eat this diet because it would promote fast accumulation of glycogen in the muscles. Also, having the subjects eat the same foods eliminated the possibility that different foods would cause variation in results.

Q: Why couldn't the men exercise for 2-3 days after the study?
A: The tests were run over a 2-3 day period after the study exercises. If the men exercised in this time period, the study results would reflect not only the accumulation of glycogen after the study exercises, but also the additional exercising.

Q: What is a catheter?
A: A catheter is used to administer substances directly into the blood stream, or to allow blood to be drawn quickly. It is a narrow, plastic tube that has a port at one end and an open tip at the other end. A needle fits inside the tube and the needle point protrudes from the open end of the tube. It is inserted into the vein and the needle is pulled out, leaving only the tube in the vein. IV lines or syringes can be attached to the tube's port, allowing substances to be released into the vein or blood taken from it very quickly. In the study, catheters allowed researchers to draw the subjects' blood for tests at more precise time intervals.