Background The first year of university has been identified as a period of adoption of unhealthy lifestyle behaviors. However, only a few studies have addressed the extent of this phenomenon in Latin American universities. The aim of this study was to examine changes in physiotherapy students' lifestyle behaviors after one year at university. Methods A total of 100 students of Universidad de Las Americas in Quito, Ecuador, were prospectively recruited. In all patients' sociodemographic variables, anthropometric indices, adherence to 24-hour movement guidelines (i.e., physical activity, sedentary behavior, and sleep), diet, and alcohol consumption were assessed. Measurements were performed at the start of the first academic semester and after one year at university. Results The total of sample, 60 students were female (60%), 40 were male (40%), and the mean age was 21.2 years old. At one-year follow-up, the mean difference (MD) for weight was +5.8 kg [95% confidence interval (CI): 2.9 to 8.5; p < 0.001] and for body mass index was +3.3 kg/m(2) (95% CI: 1.1 to 5.2; p = 0.001). Additionally, number of students with high levels of physical activity according to the Global Physical Activity Questionnaire (GPAQ) decreased from 42 to 28 (p < 0.001), the MD for total metabolic equivalents (METs) per week was -1.0 (95% CI -0.8 to -2.0; p = 0.001), and decreased from 10 to 6 students (p = 0.042) who met the overall 24-hour movement guidelines. Finally, there was an increase from 70 to 79 students (p = 0.041) who showed significant sleep disturbances, from 70 to 80 students (p = 0.035) who needed changes in diet quality, and from 20 to 30 students (p = 0.035) who showed harmful alcohol consumption. Conclusions At the end of the first year of university, most students showed unhealthy lifestyle behaviors characterized by an increase in weight and body mass index, a decrease in the level of physical activity, low adherence to 24-hour movement guidelines, more sleep disturbance, poor diet quality, and harmful alcohol consumption. This indicates a need to address this problem during this critical period for developing interventions to improve lifestyle behaviors and prevent the occurrence of non-communicable chronic diseases.