Purpose: The present study was designed to observe the concentration of hydrogen peroxide (H2O2) in exhaled breath condensate (EBC) after induced phonotrauma. Methods: Thirty-five participants were randomly assigned to one of two conditions (1) Vocal demand and (2) Control. Participants in the experimental group (vocal demand) were asked to read aloud some texts during 1 hour, at 85-90 dB. Inflammation (H2O2 from exhaled breath condensate), acoustic, aerodynamic, and subjective measures were obtained at four time points: before vocal demand (baseline), immediately after baseline, 4-hour after baseline, and 24 hours after baseline. The same acquisition process was implemented for subjects in control group, except that they were not asked to engage in any vocal demand tasks at all. Results: As for biological samples, a significant effect for group was observed. Higher values were found for participants in experimental condition. Significant differences were observed for within contrasts in the experimental group, namely 4 hours against baseline, 4 hours against immediately post, and 24 hours against 4 hours. Instrumental outcomes did not show significant differences across the different conditions at any time points. Self-reported measures (vocal fatigue and sensation of muscle tension) showed a significant main effect for group and main effect for condition. Conclusions: Intense vocal demand causes an increase in the concentration of H2O2 obtained from EBC at four hours after baseline, which is compatible with the generation of an inflammatory process in the vocal folds (phonotrauma). Moreover, the increase in the sensation of vocal fatigue and muscle tension after demand tasks seems to be an immediate reaction that did not match in time with the increment of H2O2 concentration.