We present an experimental paradigm to measure changes in characteristics of speech under vocal fatigue. For speech corpora, we have chosen a vocal load (3 hours) and a cognitive process (reading aloud continuously) that can induce some fatigue of the reader. Fatigue is verified using an analysis of reading errors and disfluencies. A multivariate analysis based on Wilks' lambda test, of 169,042 occurrences of phonemes, can analyze spectral and prosodic changes of each phonetic class. Based on six readers, the results show that nasals (vowels and consonants) are the most discriminant phonemes in vocal fatigue.