Objectives: To determine energy status of spheroids formed of NCI-H358 cell line in dependence of medium availability and sphere growth. Study Design: In vitro controlled trial. Material and Methods: NCI-H358 cell line was grown in RPMI medium and incubated in CO2 incubator after which the number of living cells was determined by erythrosin B staining. Cells were seeded on microtiter plates, centrifuged and incubated to form spheroids. Spheroids were cultivated in two time periods: 8 and 13 days where to one set of spheres medium exchange was preformed every few days, while other set was cultivated without medium exchange. Measurement of lactate dehydrogenase was preformed by measuring abosorbance at 515 nm which was used to calculate LDH quantity. To determine mitochondrial potential, spheres were trypsinized and cells were incubated with TMRE stain. The fluorescence intensity was measured at Ex/Em = 530/580. Colorimetric analysis of adenosine triphosphate was done by measuring absorbance at 515 nm which was used to calculate ATP concentration. Results: Spheres cultivated for 8 days show similar mitochondrial and LDH activity. The lowest LDH activity was measured in spheres cultivated 13 days with medium exchange, but the highest value was in spheroids cultivated in same time period without medium exchange due to necrosis. Significantly high membrane potential was shown in spheroids cultivated for 13 days with medium exchange due to viable cells activity and hipoxy stimulation. Measured ATP concentrations were extremely low. Conclusion: Medium availability affects on energy status of spheres cultivated 13 days. Hipoxia and necrosis stimulate activity of LDH and mitochondria, while membrane potental grows with the number of viable cells.