This study was conducted to measure the reliability of an indoor uphill time trial (TT) using the Velotron electronic bicycle ergometer with a computer-generated pacer that represented a subject's prior TT performance. A total of 12 trained cyclists (42 ± 7.79 years, 63.0 ± 6.12 ml/kg/min) completed three 8-mile uphill TT with 2-7 days between subsequent tests. A repeated measure ANOVA found no difference between finishing times for TT1 (2,154 ± 246 s) and TT2 (2,078 ± 185 s, p = 0.055) or TT2 and TT3 (2,047 ± 167 s, p = 0.075). All measures of reliability showed an increased reliability for finishing times in TT2-TT3 [95% CI for: coefficient of variation (CV) = 1.0-2.3%, intraclass correlation coefficient (ICC) = 0.953-0.996, standard error of measurement (SEM) = 2.1-4.9 s] versus TT1-TT2 (95% CI for: CV = 2.0-4.9%, ICC = 0.827-0.986, SEM = 9.8-23 s). An uphill TT using the Velotron bicycle ergometer with a prior performance as a pacer is a reliable assessment for indoor TT performance testing, but a familiarization trial improves reliability. Although there were no significant differences found for completion times between TT1-TT2 or TT2-TT3, the absolute mean differences, small sample size and low p values may be suggestive of a Type II error.