Spatial separation in emergency departments (ED) is empirically practised as part of transmission-based precaution. Despite its potential benefits in segregating potentially infectious patients, the effects of spatial separation on patient flow remains uncertain. To explore the impact of spatial separation on ED patient flow and identify specific clinical factors and flow process intervals (FPI) influencing ED length-of-stay (EDLOS). This was a retrospective study of data extracted from patients' electronic medical records from January 1 to March 31, 2022 conducted at the ED of a tertiary hospital in Kuala Lumpur, Malaysia. During this period, patients were separated into respiratory areas (RA) and non-respiratory areas (NRA) based on Centers for Disease Control and Prevention recommendations. The study obtained ethics approval from the institution's ethics board. A total of 1,054 patients were included in the study, 275 allocated to RA and 779 to NRA. Patients in RA had a significantly longer median EDLOS compared to in NRA (9 hours 29 minutes versus 7 hours 6 minutes, p<0.001, d=0.41). A lower proportion of patients in RA achieved an EDLOS ≤8 hours compared to NRA (41.8% versus 58.3%, p<0.001). Independent factors affecting EDLOS were triage category, re-triaging, hypertension, performing biomedical imaging, medical, surgical, and critical care consultations, and disposition plan. Bottlenecks significantly prolonging EDLOS were decision-to-departure, ultrasound interval and referral-to-consultation. Spatial separation prolongs FPI and EDLOS. Addressing inpatient access block and streamlining specialty review and biomedical imaging processes may reduce RA EDLOS.