Background and Objective:Functional connectivity (FC) measures can be used to differentiate epileptogenic zones (EZs) from non-EZs in patients with medically refractory epilepsy. However, little work has been done to evaluate stability of stereoelectroencephalography (SEEG) FC measures over time and their relationship with anti-seizure medication (ASM) use – a critical confounder in epilepsy FC studies to date. In this study, we aim to answer the following questions: 1) are SEEG FC measures stable over time, 2) are they influenced by ASMs, and 3) are they impacted by patient data collection state?Methods:In 32 patients with medically-refractory focal epilepsy, we collected a single two-minute prospective SEEG resting-state (awake, eyes closed) data set, and consecutive two-minute retrospective pseudo-rest (awake, eyes open) data sets for days 1-7 post-implantation. ASM dosages were recorded for days 1-7 post-implantation and drug load score (DLS) per day was calculated to standardize and compare across patients. FC was evaluated using directed and nondirected measures. Standard clinical interpretation of ictal SEEG was used to classify brain regions as EZs and non-EZs.Results:Over seven days, presumed EZs consistently had higher FC than non-EZs when using Between Imaginary Coherence (ImCoh) and Partial Directed Coherence (PDC) Inward strength, without accounting for DLS. These measures were demonstrated to be stable over a short-term period of three consecutive days with the same DLS. Between ImCoh FC differences between EZs and non-EZs were reduced with DLS decreases, whereas other measures were not affected by DLS. FC differences between EZs and non-EZs were seen during both resting-state and pseudo-rest conditions; ImCoh values were strongly correlated between the two conditions, whereas PDC values were not.Discussion:Inward and non-directed SEEG FC is higher in presumed EZs versus non-EZs, and measures are stable over time. However, certain measures may be impacted by ASM dose, as Between ImCoh differences between EZs and non-EZs are less pronounced with lower doses, and other measures such as PDC are poorly correlated across recording conditions. These findings allow novel insight into how SEEG FC measures may aid surgical localization, and how they are influenced by ASMs and other factors.