Time-trials (TTs) represent an ecologically valid approach to assessment of endurance performance. With no previous research available, the present study aimed to investigate the intra-subject variability of 5km TT running performance in moderately trained runners. Such information is useful in the application of testing protocols as well as in the estimation of sample sizes required for scientific research as per statistical significance and magnitude based inference methods. Six competitive moderately trained male runners completed an incremental exercise test to volitional exhaustion followed by 5 x 5 km TTs (including a familiarisation trial), individually spaced by 48 hours. Time taken to complete each trial, heart rate, rating of perceived exertion (RPE) and speed were all assessed. For the primary measure time, results showed a coefficient of variation (CV) score across all participants of 1.5% ± 0.59% with an intra-class correlation coefficient (ICC) score of 0.990. Heart rate, RPE and speed data showed a variance range between 0.8% - 3.05 (CV). It was concluded that when compared with related research, there was an observed low intra-subject variability in trained runners over a 5km distance. This supports the use of this protocol for 5km TT performance for assessment of nutritional strategies, ergogenic aids, or training interventions on endurance running performance.