Objectives The aim of this study was to evaluate the acoustic measurements of the vowel /a/ in modal recording before and after a singing voice resistance test and after 30 minutes of absolute rest in female choir singers. Study Design This is a prospective cohort study. Methods A total of 13 soprano choir singers with experience in choir singing were evaluated through analysis of acoustic voice parameters at three points in time: before continuous use of the voice, after vocal warm-up and a singing test 60 minutes in duration respecting the pauses for breathing, and after vocal cooldown and an absolute voice rest for 30 minutes. Results The fundamental frequency increased after the voice resistance test (P = 0.012) and remained elevated after the 30 minutes of voice rest (P = 0.01). The jitter decreased after the voice resistance test (P = 0.02) and after the 30 minutes of voice rest. A significant difference was detected for the acoustic voice parameters relative average perturbation (RAP), (P = 0.05), and pitch perturbation quotient (PPQ), (P = 0.04), compared with the initial time point. Conclusions The fundamental frequency increased after 60 minutes of singing and remained elevated after vocal cooldown and absolute rest for 30 minutes, proving an efficient parameter for identifying the changes inherent to voice demand during singing.