This study evaluated the impact of short-term feed restriction in lactating dairy cows on regional permeability of the gastrointestinal tract (GIT), and the recovery of DMI, ruminal pH, and milk yield. In addition, sampling methods for a novel dual marker technique to characterize total GIT and post ruminal permeability were validated. Six ruminally cannulated lactating Holstein cows were blocked by parity (3 primiparous, 3 multiparous; 189 DIM ± 25.2) and enrolled in a crossover design. Experimental periods included a 5-d baseline phase (BASE), 5-d challenge phase (CHAL), and 2 weeks of recovery (REC1 and REC2). During CHAL cows received either 100% ad libitum feed intake (AL) or 40% of ad libitum feed intake (FR). To assess, total-tract and post-ruminal permeability, equimolar doses of Cr-EDTA and Co-EDTA were infused on d 3 of CHAL into the rumen and abomasum (0.369 mmol/kg BW). Following infusions, total urine and feces were collected every 8 h over 96 h, and blood samples were collected at h 0, 1, 2, 3, 4, 6, 8, 12, 16, 20, 24, 32, 40, 48, and 64. The plasma area under the curve (AUC) for Cr and Co were calculated. By design, DMI for FR was reduced by 60% during CHAL and remained 19% lower than AL during REC1 but was not different from AL in REC2. Mean ruminal pH for FR was greatest during CHAL and the least during REC1, with no differences detected between AL and FR in REC2. The duration that pH was < 5.8 was the least for FR during CHAL and greatest during REC1 which were different from AL and were no longer different between treatments in REC2. Milk yield was the least for FR during CHAL and REC1 and no longer different from AL in REC2. Feed restriction reduced milk fat, protein, and lactose yields by 26, 31% and 31%, respectively. Plasma Cr AUC was 34% greater and Co AUC tended to be 35% greater for FR than AL on d 3 of CHAL. Urinary Cr recovery after 48-h was not affected by treatment; however, urinary Co recovery was 36% greater for FR than AL. Positive correlations between plasma AUC and urinary recovery for Cr and Co were detected. It was determined that blood samples collected at h 2, 8, 20, 40, and 48 could predict the total plasma Cr and Co AUC within 1.9% and 6.2%, respectively. In summary, short-term FR in lactating dairy cows increases permeability of the total GIT and may increase permeability of the post-ruminal regions with more than 60% of the permeability occurring post-ruminally. After FR, cows experienced low ruminal pH and a sustained reduction in milk yield. When utilizing Cr- and Co-EDTA to evaluate regional GIT permeability, plasma AUC can be used as an alternate to urinary Cr and Co excretion. In addition, blood samples collected at h 2, 8, 20, 40, and 48 result in adequate prediction accuracy, at least when comparing GIT permeability for lactating dairy cows exposed to AL and FR.