Laboratory based cycling time trials (TT) are widely used by both researchers and practitioners, as a method of assessing cycling performance in a controlled environment. Assessments of performance often use TT durations or distances between 20 min and one hour and in the UK the 10 mile (16.1 km) TT is the most frequently used race distance for trained cyclists. The 16.1 km TT has received relatively minimal, but increased attention as a performance criterion in the literature. Therefore, the aim of this study was to assess the reliability of 16.1 km TT performance in a large cohort of trained cyclists using the CompuTrainer cycling ergometer. Trained male cyclists (n = 58, mean±SD age 35±7 yr, height 179±6 cm, weight 79.1±9.4 kg, VO2max. 56.6±6.6 ml.kg.min-1, PPO 365±37 W) performed an initial incremental exercise test to determine PPO and VO2max. The participants then performed two 16.1 km TT on a CompuTrainer cycle ergometer separated by 3-7 days. Differences in time, power output and speed were determined using a Wilcoxon signed ranks or paired t-tests. Reproducibility of the TT performance measures was performed using the coefficient of variation (CV), intraclass correlations, and typical error (TE). There were no differences between any of the performance criteria for the whole cohort (Mean difference = 0.06 min, 0.09 km.h-1, 1.5 W, for time, mean speed and power respectively) between TT1 and TT2. All TT performance data were very reproducible (CV range = 1.1-2.7%) and demonstrated trivial or small TE. The slower cyclists demonstrated marginally lower reliability (CV range = 1.3-3.2%) compared to the fastest group (CV range = 0.7-2.0%). The 16.1 km TT on the CompuTrainer represents a very reliable performance criterion for trained cyclists. Interpretation of test-retest performance outcomes should be performed in the context of the TE of each performance indicator.