The aim of this study was to perform a preliminary validation of the TT5km to 1) assess changes of the aerobic endurance performance and 2) rank well-trained cyclists based on aerobic endurance fitness. The TT5km can rank the aerobic endurance fitness among well-trained cyclists due to the adequate absolute and relative reliability, as well as high correlation with aerobic endurance indices obtained from the incremental test. The TT5km can also differentiate the changes in aerobic endurance performance once the performance variables presented the smallest worthwhile change higher than typical error. When the performance variables were compared (5K
PO, 5K
AVS, 5K
Time), we found higher performance in the TT5km
2. Although the sample comprised experienced athletes in road cycling, habituated to perform time trials, such differences may be related to the absence of a specific familiarization session. However, the pacing strategy between TT5km tests (interaction effect) was not significantly different from all variables, excepting for quadriceps electromyographic activity. Although the athletes predominantly increased their performance in the TT5km
2, they maintained their pacing strategy and rank position in the classification test. This information is supported by strong correlations between performance variables from the TT5km
1 and TT5km
2, and consistency of the relative reliability data. Thus, the consistency with the TT5km
2 showed that TT5km
1 could differentiate performance of the cyclists. Further, absolute reliability of performance indices was acceptable. These arguments emerge a critical problem around this point: familiarization sessions vs. available time for the assessments. Even the learning effect being known (
2,
6,
11), multiple familiarization sessions are unfeasible in the practical environment. Thus, a test that has already presented reliability and high correlation between the two first sessions is a desirable option for practical purposes. Moreover, the TT5km presents these characteristics even when it is compared to 40-km time trial data (
11,
12). However, despite the absolute reliability of the TT5km performance variables was compatible with other time trials (
6,
11,
12), our results suggest that one familiarization session may be required, mainly when using the TT5km for the first time. More sessions are not required in theory because reliability studies using time trials have shown that performance between the second and subsequent sessions are reproduced adequately (
6). The TT5km has the coaches’ desirable characteristics (e.g. short duration, lower level of fatigue), even when the familiarization session is required. Moreover, for the subsequent re-evaluations, this familiarization session may not be required once time trials present good reliability even when repeated after a long time (
13). The reliability of the TT5km performance variables is equivalent to or better than longer time trials in the literature (
6,
11,
13,
35). Nonetheless, this observation was not consistently reproduced in the physiological variables. Although the heart rate had presented good absolute reliability, as described in other studies (
12,
13), it presented poor relative reliability. These results demonstrate the inability to rank the athletes’ responses using heart rate. We consider that the narrow range of intensities in which the TT5km is performed (± 4% of heart rate at anaerobic threshold) harms the consistency, where small variations can significantly change the position of a volunteer in the ranking. The non-significant correlation between heart rate average of the TT5km
1 and the TT5km
2 reinforces this argument. Therefore, it is not recommended to use heart rate to detect differences in aerobic endurance performance. Similar recommendation has to be followed to quadriceps electromyography due to its low absolute reliability, although some studies have been using such variables to evaluate neuromuscular function during research protocols (
15,
16) and training season (
1,
17). Thus, heart rate and quadriceps electromyography do not seem to be reliable parameters to evaluate changes in aerobic endurance performance. Physiological variables tend to show larger errors and they are not adequate to rank or predict changes in performance, at least when using time trials (
19,
38). The correlation and the sensitivity index in the TT5km performance variables were evaluated due to their high reliability. The physiological and neuromuscular variables did not show good reliability, making no sense for their additional evaluations. Our data presented strong correlations and shared variance between the TT5km performance variables and the aerobic endurance indices obtained from incremental test. The data are comparable with the correlations between the same indices and 40-km time trial performance indices (
9,
11). Additionally, these aerobic endurance indices are considered performance predictors in several endurance sports (
1,
7,
9,
10,
22). It is worth mentioning that the stronger correlations were between the 5K
PO (absolute and relative) and power output at the second heart rate variability threshold. This threshold is interpreted as the anaerobic threshold (
29), and high indices elicited at this physiological marker (e.g. average speed or power output) are considered essential for a successful endurance performance (
1,
7). Our performance data was better correlated with indices at the anaerobic threshold than 40-km time trial in a previous study (
11). The majority of the inter-volunteers variation in 5K
PO during the TT5km
1 (74%) and the TT5km
2 performances (82%) is explained by the power output at the second heart rate variability threshold. Besides strong correlations with power output at anaerobic threshold, the TT5km has also shown strong correlation and shared variance with 40-km time trial performance (
18,
22), which is considered a reliable time trial to evaluate aerobic endurance performance in the laboratory and field (
11,
18,
19). The 40-km time trial has also been frequently used to predict intensity at the maximal lactate steady state (
11,
18,
19). The TT5km has predicted the maximal lactate steady state and the 40-km time trial intensity (
23). Our present results demonstrated that the TT5km distinguishes the athletes regarding to the aerobic endurance level similarly to the 40-km time trial. Then, these results suggest that the TT5km is valid to assess aerobic endurance performance in cyclists and to rank athletes regarding to their aerobic endurance fitness as well as incremental and 40-km time trial tests do. Thus, if the TT5km is able to detect the changes induced by longitudinal interventions, it has more advantages than incremental test and 40 km-time trial, which are: higher capacity to assess the inherent technical ability of the modality than incremental test (
3,
9); the physiological demand and sensation more similar to real race than incremental test (
5,
6); the capacity to predict the intensity corresponding to the 40-km time trial and maximal lactate steady state more accurately than using incremental and constant load tests, performing a single session (
18,
22,
23,
38); duration and fatigue level lesser than in the 40 km-time trial. Hence, TT5km sensitivity was also evaluated. The TT5km presented sensitivity index > 1 for all performance variables, showing its ability to detect longitudinal changes in the performance (internal responsiveness). The cross-sectional studies with experimental interventions corroborate with our sensitivity data. For instance, some studies have used the TT5km in their research protocols to evaluate performance (
8,
16,
27). These studies have used interventions aiming to reduce the capacity of determinant systems for the aerobic endurance performance. Performing the TT5km, the performance (i.e. 5K
PO and 5K
Time) was greater and lower than our smallest worthwhile change values for effective (
8,
16) and non-effective reduction of capacity in the systems (
27), respectively. This information means that the smallest worthwhile change from our data could detect the effective and non-effective changes in aerobic endurance performance. Further, the estimated confidence interval for detecting the differences in performance presented in a previous study was similar to the smallest worthwhile change for 5K
PO and 5K
Time of our study (
24). Then, these results suggest adequate sensitivity and validity of the TT5km to differentiate aerobic endurance performance in cyclists. However, a longitudinal study should verify whether the magnitude of changes in the TT5km performance caused by long-term training is higher than the smallest worthwhile change values, and closely related to aerobic endurance indices change (i.e. external responsiveness) (
36). Moreover, although our sample comprised well-trained cyclists who had experience in time trials, the TT5km performance varied considerably between the best and worst athlete (e.g. almost equal to 2.5 minute). This range may have overestimated our relative reliability index, and future studies are needed to verify the relative reliability using elite cyclists. It is also, worth mentioning that the feedback of distance could have influenced the pacing strategy and learning effect. Although we cannot evaluate such influence in our study, feedback has not changed performance in short time trials similar to our protocol (
39). From the results of this study, coaches can use the TT5km to differentiate performance and to rank athletes through simple and affordable parameters, without endangering subsequent training sessions. For instance, using the confidence interval of the smallest worthwhile changes from this study, an increment of 17.7 W in average power output allows detecting improvements in aerobic endurance performance. On the other hand, a similar amount of decrement after high training loads can suggest a functional overreaching. Similar rationale can be followed in relative power output (± 0.3 W.kg
-1), time to complete the test (± 13.4 s), or average speed variables (± 1.0 km.h
-1). All of them are parameters provided by most of the cycle simulators and devices used to control training sessions. The TT5km is a valid test to rank the aerobic endurance fitness of well-trained cyclists and to differentiate changes on aerobic endurance performance. However, a familiarization session is recommended for the first time the test will be performed. A future study should investigate the longitudinal validity of the TT5km.