The purpose of this study is to analyze the effect of two genetic polymorphisms, ACTN3 R577X , and ACE I/D , on physical condition in a sample of active older women after a two-year training period. The sample was composed of 300 healthy women over the age of 60 who underwent a two-year training program. Adapted tests from the Senior Fitness Test were used. The genotyping of the polymorphisms was obtained from the participants’ DNA via buccal swabs. The analysis of the ACE polymorphism did not reveal differences between genotypes. The analysis of the R577X polymorphism showed a favorable effect for the ACTN3 XX genotype in tests for leg strength ( p : 0.001) after training, compared to the other genotypes, and also in the analysis of the combined effect of the polymorphism ( ACE II + ACTN3 RX/XX ). The intragroup effect revealed an improvement in arm strength for carriers of the X allele after 24 months of training ( p < 0.05). The endurance values significantly worsened in all study groups. Conclusions: The R577X polymorphism of ACTN3 may have an important role in capacities related to muscle strength, providing a beneficial effect for carriers of the X allele.