Background: Premature neonates are a group of high-risk patients who need intensive medical care and hospitalization in the neonatal intensive care unit (NICU) for their survival. In the NICU environment, there are highly stressful stimuli that have a negative effect on the physiological parameters of premature neonates. One of the interventions that improve the physiological parameters of neonates is the implementation of the quiet time protocol. Objectives: The present study aimed to investigate the effect of the quiet time protocol intervention on the physiological parameters of premature neonates admitted to the NICU. Methods: This quasi-experimental study was performed on 62 premature neonates selected through a convenient sampling method out of the neonates admitted to the NICU. First, the samples of the control group were collected. A demographic data questionnaire was employed. Neonates in the two groups were examined for 60 min during three phases before, during, and after the intervention in the evening shift of 15 - 17 o’clock. Data were analyzed using the SPSS software version 22 by the chi-squared test, repeated measures analysis of variance (ANOVA), and t-test. P-value < 0.05 was considered statistically significant. Results: The results of repeated measures ANOVA regarding arterial blood oxygen saturation (SaO2) percentage showed that changes over time were significant (P < 0.001). There was also a significant difference between the groups in terms of intervention effect (P = 0.004). In other words, the changes in SaO2 percentage were not the same in the two groups, and the changes in the score of the intervention group were higher than the control group. The results of repeated measures ANOVA showed that the changes in the heart rate and respiratory rate over time were significant (P < 0.001). Conclusions: The quiet time protocol is effective in reducing environmental stimuli and improving physiological parameters. Therefore, the implementation of these procedures during each 8-hour shift, which includes a specific period which light, sound, and bedside stimulation is controllably reduced, is recommended as a standard of care to reduce stress and improve the growth and development of premature neonates in the NICU.