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In music exhibiting two main themes, the first theme is commonly characterized as stronger or more energetic, whereas the second theme is characterized as gentler or more cantabile. Previous work indicates that compared to first themes, second themes are less likely to be in the minor mode, but are more likely to be legato, make use of a slower tempo, utilize smaller intervals, and involve a quieter dynamic level. The current study tested whether musicians are sensitive to these changes in the musical structure. Musicians (n = 44) were tested on their ability to discriminate between pairs of musical themes in 22 piano works. Participants completed two tasks, one based on the musical score and one based on sound recordings. The study was designed as a two-alternative forced-choice task, where musicians were asked to identify which of the two themes comes first in the music. Additionally, the type of information available in the musical excerpts was manipulated so that in some trials, participants did not have access to dynamic and articulative information. The results indicate that musicians are weakly able to differentiate between first and second themes in both the score-only (p < 0.001) and audio-only tasks (p < 0.001), and that participants perform better when stimuli include dynamic and articulative information (p < 0.02). The results are consistent with the claim that musicians can weakly perceive structural and expressive differences between first and second themes, even outside of the original musical context.