Six1 is a transcription factor that, along with cofactors (Eya1, Eya3, and Dach2), regulates skeletal muscle fiber-type and development. SIX1 (human) gene expression decreases after overload, but the time course of Six1 expression, if protein is affected, and if the response differs between muscles with differing phenotypes, is not known. Our purpose was to examine Six1 gene and protein expression and co-factor gene expression during the initiation of muscle overload, and determine if the muscle phenotype altered this response. The plantaris and soleus were functionally overloaded by synergistic ablation of the gastrocnemius, and Six1 gene and protein, and Six1 cofactor gene expression was measured. Six1 gene expression decreased at 1 day of overload 48 ± 9 and 47 ± 20 % (p < 0.01) in the plantaris and soleus. After 3 days of overload, Six1 protein expression increased 73 ± 17 and 168 ± 57 % in the plantaris and soleus (p < 0.05). After 1 day of overload, Dach2 gene expression decreased 56 ± 9 and 35 ± 3 % in both muscles (p < 0.001), while Eya1 decreased 33 ± 5 % only in the soleus (p < 0.01). Eya3 gene expression increased 127 ± 26 % (p < 0.05) and 76 ± 16 % (p < 0.05) in the plantaris and soleus, while Dach2 gene expression decreased 71 ± 4 % (p < 0.05) in the soleus after 3 days of overload. Six1 and Six1 co-factor expression is responsive to muscle overload in both fast and slow muscles. This indicates that this molecular program may affect overload adaptation regardless of muscle phenotype.