The first two days of introduction to treadmill running consists of simply placing the rat on the belt that is moving at a velocity of 10 m/min (15° slope) and picking the rat up and moving it forward if it starts to slide off the back of the belt. During introduction days 3-5, the belt speed is gradually increased up to 15 m/min and failure to run will cause the rats to slide off of the moving belt and onto a 15 x 15 cm electric shock grid that delivers 1.2 mA of current at 3 Hz. The rats are left on the grid for about 1.5 sec and then moved forward onto the moving belt. This process is repeated until the rats learn to run in order to avoid the mild shock. The ability to achieve this minimal level of running at least once constitutes the threshold performance necessary for inclusion in evaluation for maximal capacity the following week. Rats not achieving this minimal running capacity are dropped from further testing; i.e., the low line is selected for low running capacity, not zero running capacity. We lose about 6% of the rats at each generation because they do not complete the threshold test.

During the second week, each rat is evaluated for maximal endurance running capacity on five consecutive days. Each daily endurance trial is performed at a constant slope of 15° with the starting velocity at 10 m/min. Treadmill velocity is increased by 1 m/min every 2 min and each rat is run until exhausted. Exhaustion is operationally defined as the third time a rat can no longer keep pace with the speed of the treadmill and remains on the shock grid for two seconds rather than run. At the moment of exhaustion, the current to the grid is stopped and the rat removed from the treadmill and weighed.

For each of the five trials, the total distance run (m) is used as the estimate of aerobic endurance capacity. The single best daily run of five trials for each rat is considered the trial most closely associated with the genetic component of endurance running capacity. This idea of estimating the genetic component from the best performance, rather than (for example) the average of all trials, has two origins:

1. The environment can have an infinite negative influence upon performance (i.e., a detrimental environment can take the performance to zero). Factors such as subtle differences in housing or daily handling could cause a genetically superior rat to perform below its maximal ability on a given day.



2. However, the environment can have only a finite positive influence upon endurance running performance. That is, environmental influences cannot cause a rat to perform above the genetically determined upper limit of its ability. Thus, the rat’s best performance comes closest to the genetically determined upper limit of its ability. Because our goal was to select for genetically determined differences in phenotype, we used the best performance rather than the average.

The estimate of capacity is thus based upon the single best day of running for each rat.

The nomogram below shows the relationship between distance run, time, and speed for the ramped running protocol. Given the need to test large numbers of rats, the running protocol was designed to provide a test of primarily aerobic endurance capacity that could be completed in a reasonable time. That this goal was achieved is evidenced by the values of average capacity for rats in the founder population and after six generations of high line selection. The maximal running capacity of the founder population was 355 meters, accomplished in about 22 min at a maximal speed of 20 m/min. The sixth generation of selection in the high line produced a population that ran to exhaustion in 839 m (time = 42 min and maximum speed = 30 m/min). These running distances and times far exceed the expected capacity of rats for anaerobic exercise.

Nomogram relating speed and time to distance run to exhaustion for the speed-ramped protocol. The starting speed was 10 m/min and was increased 1 m/min every 2 minutes. Average distance run by the founder population and by the high-line rats at generation six are indicated by the vertical arrows.