Tympanometry is a physiological measurement of the acoustic admittance in sound transmission through the ear canal and middle ear and has been widely used in audiology as an objective and non-invasive means to determine the function of the middle ear system. A graphic display of the measurement is called a tympanogram. Characteristics of tympanograms have been quantified with several measures, e.g., peak compensated static acoustic admittance (Ytm) and equivalent ear canal volume (Vec). In the past decades, numerous investigations confirmed the effect of several middle-ear pathologies on tympanometric measures. However, little effort has been made to specifically explore the effect of negative middle-ear pressure. The objective of the present study was to present the outcomes of two tympanometric measures (Ytm and Vec) in human ears with negative middle ear pressure. Data was obtained from 77 patients' records (96 ears) at the Wichita State University Speech-Language-Hearing Clinic. Results demonstrate that substantial overlap exists in both Ytm and Vec measures of tympanometry between the ears with negative middle ear pressures and those with normal pressure in previous studies. These measures are unlikely useful to serve as an index in the diagnosis of negative middle ear pressure in humans. The present study also suggests that both Ytm and Vec tend to decrease with decreasing negative middle ear pressure.