Exertional heat stress adversely distrupts (GI) barrier integrity and, through subsequent microbial translocation (MT), negativly impacts health. Despite widespread application, the temporal reliability of popular GI barrier integity and MT biomarkers is poorly characterised.Fourteen males completed two 80-min exertional heat stress tests (EHST) separated by 7-14 days. Venous blood was drawn pre, immediately- and 1-hr post both EHSTs. GI barrier integrity was assessed using the serum Dual-Sugar Absorption Test (DSAT), Intestinal Fatty-Acid-Binding Protein (I-FABP) and Claudin-3 (CLDN-3). MT was assessed using plasma Lipopolysaccharide Binding Protein (LBP), total 16S bacterial DNA and Bacteroides DNA.No GI barrier integrity or MT biomarker, except absolute Bacteroides DNA, displayed systematic trial order bias (p ≥ .05). I-FABP (trial 1 = Δ 0.834 ± 0.445 ng ml ; trial 2 = Δ 0.776 ± 0.489 ng ml ) and CLDN-3 (trial 1 = Δ 0.317 ± 0.586 ng ml ; trial 2 = Δ 0.371 ± 0.508 ng ml ) were increased post-EHST (p ≤ .01). All MT biomarkers were unchanged post-EHST. Coefficient of variation and typical error of measurement post-EHST were: 11.5% and 0.004 (ratio) for the DSAT 90-min postprobe ingestion; 12.2% and 0.004 (ratio) at 150-min postprobe ingestion; 12.1% and 0.376 ng ml for I-FABP; 4.9% and 0.342 ng ml for CLDN-3; 9.2% and 0.420 µg ml for LBP; 9.5% and 0.15 pg µl for total 16S DNA; and 54.7% and 0.032 for Bacteroides/total 16S DNA ratio.Each GI barrier integrity and MT translocation biomarker, except Bacteroides/total 16S ratio, had acceptable reliability at rest and postexertional heat stress.