This study examined the validity and reliability of a sequential “Run-Bike-Run” test (RBR) in age-group triathletes. Eight Olympic distance (OD) specialists (age 30.0 ± 2.0 years, mass 75.6 ± 1.6 kg, run VO_2max 63.8 ± 1.9 ml· kg^-1· min^-1, cycle VO_2peak 56.7 ± 5.1 ml· kg^-1· min^-1) performed four trials over 10 days. Trial 1 (TRVO_2max) was an incremental treadmill running test. Trials 2 and 3 (RBR₁ and RBR₂) involved: 1) a 7-min run at 15 km· h^-1 (R₁) plus a 1-min transition to 2) cycling to fatigue (2 W· kg^-1 body mass then 30 W each 3 min); 3) 10-min cycling at 3 W· kg^-1 (B_submax); another 1-min transition and 4) a second 7-min run at 15 km· h^-1 (R₂). Trial 4 (TT) was a 30-min cycle - 20-min run time trial. No significant differences in absolute oxygen uptake (VO₂), heart rate (HR), or blood lactate concentration ([BLA]) were evidenced between RBR₁ and RBR₂. For all measured physiological variables, the limits of agreement were similar, and the mean differences were physiologically unimportant, between trials. Low levels of test-retest error (i.e. ICC <0.8, CV<10%) were observed for most (logged) measurements. However [BLA] post R₁ (ICC 0.87, CV 25.1%), [BLA] post B_submax (ICC 0.99, CV 16.31) and [BLA] post R₂ (ICC 0.51, CV 22.9%) were least reliable. These error ranges may help coaches detect real changes in training status over time. Moreover, RBR test variables can be used to predict discipline specific and overall TT performance. Cycle VO_2peak, cycle peak power output, and the change between R₁ and R₂ (deltaR₁R₂) in [BLA] were most highly related to overall TT distance (r = 0.89, p < 0. 01; r = 0.94, p < 0.02; r = 0.86, p < 0.05, respectively). The percentage of TR VO_2max at 15 km· h^-1, and deltaR₁R₂ HR, were also related to run TT distance (r = -0.83 and 0.86, both p < 0.05).