The purpose of this study was to investigate changes in total retinal blood flow (RBF) using Doppler Fourier Domain Optical Coherence Tomography (Doppler FD-OCT) in response to the manipulation of systemic partial pressure of CO2 (PETCO2). Double circular Doppler blood flow scans were captured in nine healthy individuals (mean age ± standard deviation: 27.1 ± 4.1, six males) using the RTVue(™) FD-OCT (Optovue). PETCO2 was manipulated using a custom-designed computer-controlled gas blender (RespirAct(™)) connected to a sequential gas delivery rebreathing circuit. Doppler FD-OCT measurements were captured at baseline, during stages of hypercapnia (+5/+10/+15 mmHg PETCO2), return to baseline and during stages of hypocapnia (-5/-10/-15 mmHg PETCO2). Repeated measures analysis of variance (reANOVA) and Tukey's post hoc analysis were used to compare Doppler FD-OCT measurements between the various PETCO2 levels relative to baseline. The effect of PETCO2 on TRBF was also investigated using linear regression models. The average RBF significantly increased by 15% (P < 0.0001) with an increase in PETCO2 and decreased significantly by 10% with a decrease in PETCO2 (P = 0.001). Venous velocity significantly increased by 3.11% from baseline to extreme hypercapnia (P < 0.001) and reduced significantly by 2.01% at extreme hypocapnia (P = 0.012). No significant changes were found in the average venous area measurements under hypercapnia (P = 0.36) or hypocapnia (P = 0.40). Overall, increased and decreased PETCO2 values had a significant effect on RBF outcomes (P < 0.002). In healthy individuals, altered end-tidal CO2 levels significantly changed RBF as measured by Doppler FD-OCT.