The vinasse is a liquid effluent resulting from the sugarcane processing for ethanol production. Ten to 18 L of vinasse are generated per liter of ethanol produced. It has a high polluting potential due to its organic load and solid concentration that can cause environmental damage if mismanaged, but since the 1980s it has been used for fertirrigation. Treatment by anaerobic digestion removes organic matter, solids, and as a by-product produces biogas and biofertilizer. As vinasse has low buffering power, it generates instability in the anaerobic process. In reactors, the gradual neutralization with limestone is adequate because this material is available in the rural environment, which is capable of providing alkalinity in an easy-to-use process. The same occurs when we use bovine manure as an inoculum alternative. The objective of the study was to evaluate the reactors startup to propose criteria for the anaerobic treatment of vinasse at thermophilic temperature. We used two 15-liter useful-volume UASB reactors, each inoculated with fresh sieved manure, in addition to 16 kg of limestone as fixed bed and alkalizing agent. The reactors acclimatization happened in three temperature ranges: (i) ambient (26 +/- 2 degrees C); (ii) mesophilic (36 +/- 2 degrees C), and thermophilic (52 +/- 2 degrees C). We evaluated efficiency by the biogas yield generated and the removal of COD, when the temperature increased from 26 to 52 degrees C and the organic load from 0.02 to 1.70 gCOD.L-1.d-1. We quantified solids (Totals and Volatile Totals), pH, total alkalinity, and volatile acidity. Results showed that diluted manure produced 0.11 Lgas.gConsumedCOD-1 at 48 days of the stabilization phase, which lasted 72 days, ending with the sludge COD at 8.4 gO2.L-1. During the inoculum acclimatization, HRT was adjusted to 4 days, maximum vinasse organic load to 1.70 gCOD.L-1, and total alkalinity/volatile acidity ratio varied from 0.14 to 0.86; pH remained between 6.6 and 8.3. This demonstrates the reactors' capacity to withstand load variation and temperature increase. In the same period, the average gas production was 0.13 Lgas.gConsumedCOD-1, which is considered adequate for the startup.