The BOLD functional MRI (fMRI) signal is confounded by intrinsic vascular conditions and effects, but the size of the vascular effect on resting-state fMRI (rs-fMRI) functional connectivity is unknown [1]. This issue can be compounded by comorbid vascular pathologies, which may affect essential vascular properties such as cerebrovascular reactivity (CVR). CVR is typically measured as the degree of vasodilatation in response to moderate changes in end-tidal pressure of carbon dioxide (PETCO₂) [2]. In this study, we modulated CVR by varying the within-subject basal PETCO₂. Subsequently we investigate the association between CVR and rs-fMRI functional connectivity. We chose the motor network due to its simplicity and robustness [3].Eighteen healthy adults were imaged using a Siemens TIM Trio 3T MRI scanner with a 32-channel head coil. Within each subject, vascular tension was modulated by inducing different capnic conditions (separated by 4 mmHg PETCO₂) using a RespirAct breathing circuit (Thornhill Research, Toronto, ON). For each capnic condition, functional connectivity was measured using gradient-echo EPI (TR = 380 ms, TE = 30 ms, FA = 40°, 7 slices, ~3.44×3.44×6 mm3, 950 volumes). Also, at each capnic condition, CVR was assessed using a sinusoidal modulation of PETCO₂ (period = 120 s, baseline-to-peak amplitude = ±4mmHg) [4]. The BOLD data for CVR quantification were collected using a dual-echo pCASL sequence (TR = 3500 ms, TE_BOLD = 25 ms, FA = 90°, 20 slices, ~3.44×3.44×6 m3, 120 volumes). As respiratory tasks could potential influence neuronal function of motor regions controlling the respiratory system, we focused on the hand-related motor region alone, by overlapping the motor region of interest (ROI) with the activation t-map of a 2-minute motor scout (bilateral finger tapping). This functional ROI was then overlaid with the FreeSurfer per-subject segmentation of the primary motor region to remove non-motor regions. To investigate if the association between CVR and rs-fMRI connectivity is network-dependent, we also examined the executive control network (ECN). An ECN functional template [5] is overlaid with each individual brain mask, and the resulting ROI was used to define the regional average CVR and functional connectivity values in all subjects. The rs-fMRI preprocessing included motion correction, brain extraction, spatial smoothing (6mm FWHM), band-pass filtering (0.01 to 0.1 Hz), and regression of six motion parameters along with white matter and CSF average signals. Connectivity was computed using a seed-based correlation approach. Motor regions, as identified before, are used as seeds for motor network connectivity analysis, whereas for ECN two sphere seeds (radius of 6mm) is created in each hemisphere over superior-frontal and superior-parietal of the network. CVR was computed using a linear model fit of BOLD signal change to PETCO₂ change. The relationship between connectivity and CVR was assessed using a total least-squares linear fitting algorithm that takes into account measurement uncertainties on the x- and the y-axis [6]. Outliers were identified based on Cook’s distance from the initial linear model fit. Two subjects were excluded due to excessive head motion. The corresponding group-average CVR (left panel) and functional connectivity in the motor network (right panel) show that higher CVR (found at normocapnic baseline) is associated with higher rs-fMRI functional connectivity measurements, potentially attributable to nonlinearity in the vascular response to CO₂ [7]. A similar association was also found significant in the ECN. As illustrated in more detail in the scatter plot, a positive association was found between measured functional connectivity and CVR across all subjects and all three capnic conditions (p<0.05) in both motor network and ECN. Similar trends exist within each capnic condition, even though it is not significant in the ECN.In this study, we observed significant positive associations between resting-state functional connectivity and CVR. This is unlikely to be due to changes in brain function. It is unlikely to be caused by metabolic effects of CO₂, as we used mild CO₂ challenges, and both hypo- and hypercapnia resulted in similar CVR changes. This study cautions against the interpretation of rs-fMRI connectivity as a direct indicator of brain function, and stress the importance of vascular measurements to neutralize biases. This work provides the basis for further experimentation concerning the nature of vascular effects on resting-state fMRI connectivity.