Tempe has been known to have excellent nutritional properties. Few studies have been done to assess factors affecting changes of functional properties. This research was aimed to understand the effects of different fermentation time in the changes of functional properties of tempe using two different types of tempe starter culture and two different sample treatment. Tempe powder was prepared from inoculating soybean cotyledons with Rhizopus sp. molds and fermented in 4 different times (24, 36, 48, and 96 hours) which then dried and grinded into powder to be used for analyses. Functional properties of tempe including protein, protein digestibility, amino acid profiles, moisture content, total phenolic concentration, radical scavenging activity, and degree of lightness were observed. Protein content is increasing over fermentation time with the highest protein content is produced by cooked Raprima tempe fermented for 96 hours at 48.54 ± 0.95. Leucine makes the highest essential amino acids found in tempe. Glutamic acid makes the highest amino acid, while cystine, methionine, and tryptophan make the least amino acids found in tempe. Uncooked tempe made from KOPTI and Raprima fermented for 96 hours resulted in the highest total phenolic concentrations with 441.7635 ± 8.35 μgGAE/mL and 464.81 ± 20.62 μgGAE/mL at 10000 ppm respectively. Highest radical scavenging activity is present in uncooked Raprima tempe fermented for 96 hours at 38.29 ± 0.71 % at 5000 ppm. Degree of lightness is decreasing over fermentation time, with cooked tempe powder samples showing lighter appearance compared to uncooked tempe powder samples.