Aging influences the laryngeal anatomy and physiology, leading to altered vocal quality. In middle-aged women, the voice is affected by a combination of aging and menopausal transition. However, in many studies about vocal aging, the menopausal transition is not taken into account. The purpose of this study was to measure and describe the effect of aging on the vocal characteristics by comparing young women (between 20 and 28 years) and middle-aged premenopausal women (between 45 and 52 years). To determine the vocal characteristics in both groups, objective (aerodynamic measurements, vocal performance measurements, acoustic analysis, and a determination of the Dysphonia Severity Index) and subjective assessment techniques (perceptual evaluation, videostroboscopic evaluation, and Voice Handicap Index) were used. The middle-aged premenopausal women showed a smaller frequency and intensity range, a lower habitual fundamental frequency, and a higher soft phonation index compared with the young women. The results of this study are important when studying the voices of middle-aged women. Vocal characteristics of middle-aged women differ from young women, and these changes cannot only be because of hormonal changes during the menopause.