Extensive research on the oxidation of organic matter components using long-term BOD experiments at 20 and 10 °C was conducted on Lake Onego in different seasons in 2013–2017. Water samples were taken from the central part of the lake, Pukhtа, Kondopoga, Petrozavodsk Bays, as well as the estuarine zone of the River Shuya in two variations: surface horizon (0.5 to 1 m) or integral (from surface to bottom). The samples showed a multistage BOD development with two or three О2 demand stages in the first, second and linear stages, which demonstrate the different oxidation patterns of organic matter components. It was found that the type of the first stage (E- or А-type) and the water sampling procedure defined the variations of BOD parameters. As a result, four alternative effects of these factors were identified. Minimal О2 demand over the experimental period as well as a higher lability of oxidizable organic matter in the first BOD stage were found in water from the central part of the lake, confirming high quality of the water in this part of the lake. Elevated lability of oxidizable organic matter components in water from other parts of the lake was identified in spring. Strong correlation was found between various organic matter components (labile and oxidation-”stable” humic substances) and kinetic BOD parameters (R2 = 0.60–1.00). Suspended particulate matter also proved to influence BOD kinetic parameters.