Intervention programs are often sought to strengthen the Achilles tendon (AT) due to its high injury rate. Long rest periods between loading cycles have been found to increase collagen synthesis by tenocytes, suggesting rest duration may be important for tendon adaptation in vivo; however, exercise programs comparing long and short rest duration have not been directly compared. Fourteen adults completed a 12-week progressive training intervention; training sessions consisted of 5×10 isometric plantarflexion contractions each of 3-s duration performed at 90% of MVC three times weekly. Each leg was randomly allocated to long (LRT, 10-s rest) or short rest training (SRT, 3-s rest). We hypothesized that the leg allocated to LRT would demonstrate superior AT collagen organization compared to the leg receiving SRT, which would be related to improved biomechanical function. AT collagen organization and morphology were measured using ultrasound tissue characterization. AT properties were assessed before and after the intervention using a combination of dynamometry, ultrasound imaging, EMG, and motion capture. Contrary to our hypothesis, collagen organization did not improve following either training protocol; conversely, an unexpected decrease in echotype I proportion was seen after SRT (P<.001) but not LRT (P=.58), indicating an apparent protective effect of rest on collagen organization during isometric training. In contrast, AT adaptation was not appreciably enhanced by increasing intercycle rest duration; both protocols were equally effective at inducing significant strength gains and AT mechanical and material adaptation (P≤.001). Further research is necessary to identify optimal loading characteristics for injury prevention and rehabilitation.