Resting state (RS) functional connectivity (FC) MRI (rs-fcMRI) has been widely used to study functional connections between regions in the brain, and functionally connected areas across the visual cortex have been well recognized [1, 2]. Certain patterns, particularly striped patterns with spatial frequency (SF) of around 3 cycles per degree (cpd), can induce perceptual illusions/distortions and visual discomfort in most people, headaches in patients with migraine, and seizures in patients with photosensitive epilepsy [3]. The effects of these stressful patterns on visual cortical activation have been investigated [4], but their effects on visual cortical FC remain to be studied. In this preliminary study, we investigate visual stimulation effect on human visual cortical FC.Twelve healthy subjects (7 male, ages from 19 to 55) participated in the study. Each subject had four consecutive 12-min rs-fcMRI scans under four conditions: (1) eyes fixated on a fixation mark; (2) and (3) visual stimulation with alternating 2-sec stimulation on-and-off for 25 min; and (4) same as (1). The stimulus was a black-and-white striped pattern with SF 2.8 cpd. The fMRI images were acquired on a GE 3T scanner with an 8-channel head coil (TR/TE=2500/28 ms, FOV=22 cm, Matrix=64x64, slice thickness=3 mm). To functionally localize V1, each subject also had a visual cortical activation scan (200 sec) with a paradigm of five alternating 20-sec stimulation on-and-off blocks. High-resolution T1-weighted images were acquired. The visual cortical activation maps from the localizer scan were used to determine a putative V1 seed on each hemisphere, and then the left and right seeds were combined together to form a V1 seed for each subject. In RS analysis, after pre-processing RS time series in AFNI, the correlation between the mean time course of this combined V1 seed and the time course of every voxel in the brain was calculated [5-7]. Both ROI and whole brain group analyses were conducted. Fisher Z transformation was applied to the R values before statistical analyses. For ROI analysis in native space, for each of the four RS conditions, six FC maps were computed at six levels of significance: (1) P=1.0x10^-5; (2) P=1.0x10^-6; (3) P=1.0x10^-7; (4) P=1.0x10^-8; (5) P=1.0x10^-9; and (6) P=1.0x10^-10. For each significance level, the four FC maps were first used to determine a joint ROI mask in the visual cortex across all the four RS conditions, and then the mask was used to obtain a ROI-mean R value for each RS condition. Then, for each of the six P values, the group-mean of the ROI-mean R values of the 12 subjects was computed and analyzed to investigate the visual stimulation effect on the FC in the visual cortex. In whole-brain group analyses, the correlation maps of all subjects were warped to the ICBM 452 template. Whole-brain ANOVAs were carried to compare the Z values between different RS conditions.The visual stimulation effect on the FC in the visual cortex is illustrated in Fig. 1. The size of the ROI mask in the visual cortex varied with the significance level. The maximal mean mask size was 42.8 cm³ (P=1.0x10^-5) and the minimal mean mask size was 25.1 cm³ (P=1.0x10^-10). The ROI analysis showed that the stimulation significantly increased the R values in comparison to the baseline R values for all the six significance levels (paired t-test, max P=0.0053) (Fig. 2). This enhancement increased during the second half of the 25-min stimulation, and then decreased after the stimulation. After the cessation of the visual stimulation, the visual cortex FC remained significantly larger than that in the baseline for all the six significance levels (paired t-test, max P=0.0061), demonstrating a lasting enhancement effect. As shown in Fig. 2, the similar FC behavior for all the six significance levels demonstrates a stimulation-enhanced FC across the visual cortex. Besides within the visual cortex, the whole-brain ANOVAs also demonstrated alterations on the FC to V1 in other regions, such as the prefrontal cortex (Fig. 3).The 25-min visual stimulation with the stressful pattern significantly enhanced the FC within the visual cortex and altered the FC to V1 in other regions too, with a lasting effect even after the cessation of the stimulation. This suggests prolonged visual stimulation with stressful patterns may alter visual system FC network and its relationship with other networks.