The second variant, rs1815739 (c.1729C>T [p.Arg577Ter]), is a natural knockout variant in ACTN3 and has been associated with elite athletic performance.33 The derived T allele is present at a very high frequency (93%) in the Kalash. The average frequency in the remaining Pakistani populations is 47%. Using this (47%) frequency as a starting point for the forward-time simulations, we found that the very high frequency for this variant in the Kalash cannot be explained by genetic drift alone, even after 500 generations (Figure 4B). A selection signal (selection coefficient s = 0.01) achieved the observed frequency in 80% of the simulations after 500 generations. Both of these results support the long-standing isolation of the Kalash.